Compositions and methods for enhancing drug delivery across and into epithelial tissues

ABSTRACT

This invention provides compositions and methods for enhancing delivery of drugs and other agents across epithelial tissues, including the skin, gastrointestinal tract, pulmonary epithelium, ocular tissues and the like. The compositions and methods are also useful for delivery across endothelial tissues, including the blood brain barrier. The compositions and methods employ a delivery enhancing transporter that has sufficient guanidino or amidino sidechain moieties to enhance delivery of a compound conjugated to the reagent across one or more layers of the tissue, compared to the non-conjugated compound. The delivery-enhancing polymers include, for example, poly-arginine molecules that are preferably between about 6 and 25 residues in length.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 09/648,400, which claims priority to U.S.Provisional Patent Application No. 60/150,510, filed Aug. 24, 1999. Bothof these applications are incorporated herein by reference for allpurposes

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention pertains to the field of compositions and methodsthat enhance the delivery of drugs and other compounds across the dermaland epithelial membranes, including, for example, skin, thegastrointestinal epithelium and the bronchial epithelium.

[0004] 2. Background

[0005] Transdermal or transmucosal drug delivery is an attractive routeof drug delivery for several reasons. Gastrointestinal drug degradationand the hepatic first-pass effect are avoided. In addition, transdermaland transmucosal drug delivery is well-suited to controlled, sustaineddelivery (see, e.g., Elias, In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, Bronaugh & Maibach, Eds., pp 1-12,Marcel Dekker, New York, 1989.). For many applications, traditionalmethods of administering drugs are not optimal because of the very largeinitial concentration of the drug. Transdermal delivery could allow amore uniform, slower rate of delivery of a drug. Moreover, patientcompliance is encouraged because such delivery methods are easy to use,comfortable, convenient and non-invasive.

[0006] These advantages of transdermal and transmucosal delivery havenot led to many clinical applications because of the low permeability ofepithelial membranes, the skin in particular, to drugs. The difficultiesin delivering drugs across the skin result from the barrier property ofskin. Skin is a structurally complex thick membrane that represents thebody's border to the external hostile environment. The skin is composedof the epidermis, the dermis, the hypodermis, and the adenexalstructures (epidermal appendages). The epidermis, the outermostepithelial tissue of the skin, is itself composed of several layers—thestratum corneum, the stratum granulosum, the stratum spinosum, and thestratum basale.

[0007] Compounds that move from the environment into and through intactskin must first penetrate the stratum corneum, the outermost layer ofskin, which is compact and highly keratinized. The stratum corneum iscomposed of several layers of keratin-filled skin cells that are tightlybound together by a “glue” composed of cholesterol and fatty acids. Thethickness of the stratum corneum varies depending upon body location. Itis the presence of stratum corneum that results in the impermeability ofthe skin to pharmaceutical agents. The stratum corneum is formednaturally by cells migrating from the basal layer toward the skinsurface where they are eventually sloughed off. As the cells progresstoward the surface, they become progressively more dehydrated andkeratinized. The penetration across the stratum corneum layer isgenerally the rate-limiting step of drug permeation across skin. See,e.g., Flynn, G. L., In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, supra, at pages 27-53.

[0008] After penetration through the stratum corneum layer, systemicallyacting drug molecules then must pass into and through the epidermis, thedermis, and finally through the capillary walls of the bloodstream. Theepidermis, which lies under the stratum corneum, is composed of threelayers. The outermost of these layers is the stratum granulosum, whichlies adjacent to the stratum corneum, is composed of cells that aredifferentiated from basal cells and keratinocytes, which make up theunderlying layers. having acquired additional keratin and a moreflattened shape. The cells of this layer of the epidermis, which containgranules that are composed largely of the protein filaggrin. Thisprotein is believed to bind to the keratin filaments to form the keratincomplex. The cells also synthesize lipids that function as a “cement” tohold the cells together. The epidermis, in particular the stratumgranulosum, contains enzymes such as aminopeptidases.

[0009] The next-outermost layer of the epidermis is the stratumspinosum, the principal cells of which are keratinocytes, which arederived from basal cells that comprise the basal cell layer. Langerhanscells, which are also found in the stratum spinosum, areantigen-presenting cells and thus are involved in the mounting of animmune response against antigens that pass into the skin. The cells ofthis layer are generally involved in contact sensitivity dermatitis.

[0010] The innermost epidermal layer is the stratum basale, or basalcell layer, which consists of one cell layer of cuboidal cells that areattached by hemi-desmosomes to a thin basement membrane which separatesthe basal cell layer from the underlying dermis. The cells of the basallayer are relatively undifferentiated, proliferating cells that serve asa progenitor of the outer layers of the epidermis. The basal cell layerincludes, in addition to the basal cells, melanocytes.

[0011] The dermis is found under the epidermis, which is separated fromthe dermis by a basement membrane that consists of interlocking reteridges and dermal papillae. The dermis itself is composed of two layers,the papillary dermis and the reticular dermis. The dermis consists offibroblasts, histiocytes, endothelial cells, perivascular macrophagesand dendritic cells, mast cells, smooth muscle cells, and cells ofperipheral nerves and their end-organ receptors. The dermis alsoincludes fibrous materials such as collagen and reticulin, as well as aground substance (principally glycosaminoglycans, including hyaluronicacid, chondroitin sulfate, and dermatan sulfate).

[0012] Several methods have been proposed to enhance transdermaltransport of drugs. For example, chemical enhancers (Burnette, R. R. InDevelopmental Issues and Research Initiatives; Hadgraft J., Ed., MarcelDekker: 1989; pp. 247-288), iontophoresis, and others have been used.However, in spite of the more than thirty years of research that hasgone into delivery of drugs across the skin in particular, fewer than adozen drugs are now available for transdermal administration in, forexample, skin patches.

[0013] Transport of drugs and other molecules across the blood-brainbarrier is also problematic. The brain capillaries that make up theblood-brain barrier are composed of endothelial cells that form tightjunctions between themselves (Goldstein et al., Scientific American255:74-83 (1986); Pardridge, W. M., Endocrin. Rev. 7: 314-330 (1986)).The endothelial cells and the tight intercellular junctions that jointhe cells form a barrier against the passive movement of many moleculesfrom the blood to the brain. The endothelial cells of the blood-brainbarrier have few pinocytotic vesicles, which in other tissues can allowsomewhat unselective transport across the capillary wall. Nor is theblood-brain barrier interrupted by continuous gaps or channels that runthrough the cells, thus allowing for unrestrained passage of drugs andother molecules.

[0014] Thus, a need exists for improved reagents and methods forenhancing delivery of compounds, including drugs, across epithelialtissues and endothelial tissues such as the skin, the gastrointestinaltract, the eye and the blood-brain barrier. The present inventionfulfills this and other needs.

SUMMARY OF THE INVENTION

[0015] The present invention provides methods of targeting a compound toa gastrointestinal epithelium of an animal. The methods involveadministering to the gastrointestinal epithelium a conjugate thatincludes the compound and a delivery-enhancing transporter. Thedelivery-enhancing transporters, which are also provided by theinvention, have sufficient guanidino or amidino moieties to increasedelivery of the conjugate into the gastrointestinal epithelium or oculartissues compared to delivery of the compound in the absence of thedelivery-enhancing transporter. In some embodiments, delivery of theconjugate into the gastrointestinal epithelium or ocular tissue isincreased at least two-fold compared to delivery of the compound in theabsence of the delivery-enhancing transporter. In more preferredembodiments, delivery of the conjugate into the gastrointestinalepithelium is increased at least ten-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter. Thedelivery-enhancing transporter and the compound are typically attachedthrough a linker. In addition, the conjugate can comprise two or moredelivery-enhancing transporters linked to the compound.

[0016] Typically, the delivery-enhancing transporters comprise fewerthan 50 subunits and comprise at least 6 guanidino or amidino moieties.In some embodiments, the subunits are amino acids. In some embodiments,the delivery-enhancing transporters have from 6 to 25 guanidino oramidino moieties, and more preferably between 7 and 15 guanidinomoieties and still more preferably, at least six contiguous guanidinoand/or amidino moieties. In some embodiments, the delivery-enhancingtransporters consist essentially of 5 to 50 subunits, at least 50 ofwhich comprise guanidino or amidino residues. In some of theseembodiments, the subunits are natural or non-natural amino acids. Forexample, in some embodiments, the delivery-enhancing transportercomprises 5 to 25 arginine residues or analogs thereof. For example, thetransporter can comprise seven contiguous D-arginines.

[0017] In some embodiments, the delivery-enhancing transporter comprises7-15 arginine residues or analogs of arginine. The delivery-enhancingtransporter can have at least one arginine that is a D-arginine and insome embodiments, all arginines are D-arginine. In some embodiments, atleast 70% of the amino acids are arginines or arginine analogs. In someembodiments, the delivery-enhancing transporter comprises at least 5contiguous arginines or arginine analogs. The delivery-enhancingtransporters can comprise non-peptide backbones. In addition, in someaspects, the transporter is not attached to an amino acid sequence towhich the delivery-enhancing molecule is attached in a naturallyoccurring protein.

[0018] The delivery-enhancing transporters and methods of the inventionare useful for delivering drugs, diagnostic agents, and other compoundsof interest to the gastrointestinal epithelium. The methods andcompositions of the invention can be used not only to deliver thecompounds to the particular site of administration, but also providesystemic delivery. In some embodiments, the conjugate is administeredbucally or as a suppository. The compounds of the conjugate can be atherapeutic for a disease such as inflammatory bowel disease, coloncancer, ulcerative colitis, gastrointestinal ulcers, constipation andimbalance of salt and water absorption. Thus, the compounds can includeimmunosuppressives, ascomycins, corticosteroids, laxatives, antibioticsor anti-neoplastic agents. In some aspects of the invention, thecompound is targeted to the iliem and/or colon.

[0019] The delivery-enhancing transporters and methods of the inventionare useful for delivering drugs, diagnostic agents, and other compoundsof interest to the eye and other ocular tissues. In some embodiments,the conjugate is administered as eye drops or as an injection. Thecompounds of the conjugate include therapeutics for a disease such asconjunctivitis, bacterial infections, viral infections, dry eye andglaucoma. Thus, the compounds can include antibacterial compounds,antiviral compounds, cyclosporin, ascomycins and corticosteroids.

[0020] As discussed above, the compound to be delivered can be connectedto the delivery-enhancing transporter by a linker. In some embodiments,the linker is a releasable linker which releases the compound, inbiologically active form, from the delivery-enhancing transporter afterthe compound has passed into and/or through one or more layers of theepithelial and/or endothelial tissue. In some embodiments, the compoundis released from the linker by solvent-mediated cleavage. The conjugateis, in some embodiments, substantially stable at acidic pH but thecompound is substantially released from the delivery-enhancingtransporter at physiological pH. In some embodiments, the half-life ofthe conjugate is between 5 minutes and 24 hours upon contact with theskin or other epithelial or endothelial tissue. For example, thehalf-life can be between 30 minutes and 2 hours upon contact with theskin or other epithelial or endothelial tissue.

[0021] Examples of conjugate structures of the invention include thosehaving structures such as 3, 4, or 5, as follows:

[0022] wherein R¹ comprises the compound; X is a linkage formed betweena functional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R² is hydrogen, alkyl, aryl, acyl, orallyl; R³ comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶or CR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n 1 is1 to 10.

[0023] Preferably, X is selected from the group consisting of —C(O)O—,—C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—,—SONH—, phosphate, phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸are each independently selected from the group consisting of H andalkyl. In some embodiments, R₄ is S; R₅ is NHR₆; and R₆ is hydrogen,methyl, allyl, butyl or phenyl. In some embodiments, R₂ is benzyl; k, m,and n are each 1, and X is O. In some embodiments, the conjugatecomprises structure 3, Y is N, and R² is methyl, ethyl, propyl, butyl,allyl, benzyl or phenyl. In some embodiments, R² is benzyl; k, m, and nare each 1, and X is —OC(O)—. In some embodiments, the conjugatecomprises structure 4; R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen, methyl,allyl, butyl or phenyl. In some embodiments, the conjugate comprisesstructure 4; R⁵ is NHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl;and k and m are each 1.

[0024] The invention also provides conjugates in which the release ofthe linker from the biological agent involves a first, rate-limitingintramolecular reaction, followed by a faster intramolecular reactionthat results in release of the linker. The rate-limiting reaction can,by appropriate choice of substituents of the linker, be made to bestable at a pH that is higher or lower than physiological pH. However,once the conjugate has passed into and across one or more layers of anepithelial or endothelial tissue, the linker will be cleaved from theagent. An example of a compound that has this type of linker isstructure 6, as follows:

[0025] wherein R¹ comprises the compound; X is a linkage formed betweena functional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or para configuration, which aryl group can besubstituted or unsubstituted; R³ comprises the delivery-enhancingtransporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and 2.

[0026] In some embodiments, X is selected from the group consisting of—C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—,—SO₂NH—, —SONH—, phosphate, phosphonate phosphinate, and CR⁷R⁸, whereinR⁷ and R⁸ are each independently selected from the group consisting of Hand alkyl. In some embodiments, R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen,methyl, allyl, butyl or phenyl.

[0027] In preferred embodiments, the compositions of the inventioncomprise a linker susceptible to solvent-mediated cleavage. For example,a preferred linker is substantially stable at acidic pH but issubstantially cleaved at physiological pH.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1 shows a reaction scheme for the preparation of anα-chloroacetyl cyclosporin A derivative.

[0029]FIG. 2 shows a general procedure for the coupling ofcysteine-containing peptides to the α-chloro acetyl cyclosporin Aderivative.

[0030]FIG. 3 shows a reaction scheme for the coupling of the cyclosporinA derivative to a biotin-labeled peptide.

[0031]FIG. 4 shows a reaction scheme for coupling of a cyclosporin Aderivative to an unlabeled peptide.

[0032] FIGS. 5 A-H show various types of cleavable linkers that can beused to link a delivery-enhancing transporter to a biologically activeagent or other molecule of interest. FIG. 5A shows an example of adisulfide linkage. FIG. 5B shows a photocleavable linker which iscleaved upon exposure to electromagnetic radiation. FIG. 5C shows amodified lysyl residue used as a cleavable linker. FIG. 5D shows aconjugate in which the delivery-enhancing transporter T is linked to the2′-oxygen of the anticancer agent, paclitaxel. The linking moietyincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.FIG. 5E a linkage of a delivery-enhancing transporter to a biologicallyactive agent, e.g., paclitaxel, by an aminoalkyl carboxylic acid; alinker amino group is joined to a delivery-enhancing transporter by anamide linkage and to a paclitaxel moiety by an ester linkage.

[0033]FIGS. 5F and G show chemical structures and conventional numberingof constituent backbone atoms for paclitaxel and “TAXOTERE™” (R′=H,R″=BOC).

[0034]FIG. 5G shows the general chemical structure and ring atomnumbering for taxoid compounds.

[0035]FIG. 6 displays a synthetic scheme for a chemical conjugatebetween a heptamer of L-arginine and cyclosporin A (panel A) and its pHdependent chemical release (panel B). The α-chloro ester (6i) wastreated with benzylamine in the presence of sodium iodide to effectsubstitution, giving the secondary amine (6ii). Amine (6ii) was treatedwith anhydride (6) and the resultant crude acid (6iii) was converted toits corresponding NHS ester (6iv). Ester (6iv) was then coupled with theamino terminus of hepta-L-arginine, giving the N-Boc protected CsAconjugate (6v). Finally, removal of the Boc protecting group with formicacid afforded the conjugate (6vi) as its octatrifluoroacetate salt afterHPLC purification.

[0036]FIG. 7 displays inhibition of inflammation in murine contactdermatitis by releasable R7 CsA. Balb/c (6-7 weeks) mice were painted onthe abdomen with 100 μl of 0.7% DNFB in acetone olive oil (95:5). Threedays later both ears of the animals were restimulated with 0.5% DNFB inacetone. Mice were treated one, five, and twenty hours afterrestimulation with either vehicle alone, 1% R7 peptide alone, 1% CsA, 1%nonreleasable R7 CsA, 0.01%/0.1%/1.0% releasable R7 CsA, and thefluorinated steroid positive control 0.1% triamcinolone acetonide. Earinflammation was measured 24 hours after restimulation using a springloaded caliper. The percent reduction of inflammation was calculatedusing the following formula (t−n)/(u−n), where t=thickness of thetreated ear, n=the thickness of a normal untreated ear, and u=thicknessof an inflamed ear without any treatment. N=20 animals in each group.

[0037]FIG. 8 shows a procedure for the preparation of acopper-diethylene-triaminepentaacetic acid complex (Cu-DTPA).

[0038]FIG. 9 shows a procedure for linking the Cu-DTPA to a transporterthrough an aminocaproic acid.

[0039]FIG. 10 shows a reaction for the acylation of hydrocortisone withchloroacetic anhydride.

[0040]FIG. 11 shows a reaction for linking the acylated hydrocortisoneto a transporter.

[0041]FIG. 12 shows a reaction for preparation of C-2′ derivatives oftaxol.

[0042]FIG. 13 shows a schematic of a reaction for coupling of a taxolderivative to a biotin-labeled peptide.

[0043]FIG. 14 shows a reaction for coupling of an unlabeled peptide to aC-2′ derivative of taxol.

[0044] FIGS. 15A-C shows a reaction scheme for the formation of otherC-2′ taxol-peptide conjugates.

[0045]FIG. 16 shows a general strategy for synthesis of a conjugate inwhich a drug or other biological agent is linked to a delivery-enhancingtransporter by a pH-releasable linker.

[0046]FIG. 17 shows a schematic diagram of a protocol for synthesizing ataxol 2′-chloroacetyl derivative.

[0047]FIG. 18 shows a strategy by which a taxol 2′-chloroacetylderivative is linked to an arginine heptamer delivery-enhancingtransporter.

[0048]FIG. 19 shows three additional taxol-r7 conjugates that can bemade using the reaction conditions illustrated in FIG. 18.

[0049]FIG. 20 shows the results of a 3 day MTT cytotoxicity assay usingtaxol and two different linkers.

[0050]FIG. 21 shows the FACS cellular uptake assay of truncated analogsof Tat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆ (Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅(Fl-ahx-RKKRRQR), Tat₅₀₋₅₇ (Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇(Fl-ahx-KRRQRRR). Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptides for 15 min at 23° C.

[0051]FIG. 22 shows FACS cellular uptake assay of alanine-substitutedanalogs of Tat₄₉₋₅₇: A-49 (Fl-ahx-AKKRRQRRR), A-50 (Fl-ahx-RAKRRQRRR),A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR), A-53(Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARR), A-55 (Fl-ahx-RKKRRQARR),A-56 (Fl-ahx-RKKRRQRAR), and A-57 (Fl-ahx-RKKRRQRRA). Jurkat cells wereincubated with varying concentrations (12.5 μM shown) of peptides for 12min at 23° C.

[0052]FIG. 23 shows the FACS cellular uptake assay of d- andretro-isomers of Tat₄₉₋₅₇: d-Tat49-57 (Fl-ahx-rkkrrqrrr), Tat57-49(Fl-ahx-RRRQRRKKR), and d-Tat57-49 (Fl-ahx-rrrqrrkkr). Jurkat cells wereincubated with varying concentrations (12.5 μM shown) of peptides for 15min at 23° C.

[0053]FIG. 24 shows the FACS cellular uptake of a series of arginineoligomers and Tat₄₉₋₅₇: R5 (Fl-ahx-RRRRR), R6 (Fl-ahx-RRRRRR), R7(Fl-ahx-RRRRRRR), R8 (Fl-ahx-RRRRRRRR), R9 (Fl-ahx-RRRRRRR), r5(Fl-ahx-rrrrr), r6 (Fl-ahx-rrrrrr), r7 (Fl-ahx-rrrrrrr), r8(Fl-ahx-rrrrrrrr), r9 (Fl-ahx-rrrrrrrrr). Jurkat cells were incubatedwith varying concentrations (12.5 μM shown) of peptides for 4 min at 23°C.

[0054]FIG. 25 displayes the preparation of guanidine-substitutedpeptoids.

[0055]FIG. 26 displays the FACS cellular uptake of polyguanidinepeptoids and d-arginine oligomers. Jurkat cells were incubated withvarying concentrations (12.5 μM shown) of peptoids and peptides for 4min at 23° C.

[0056]FIG. 27 displays the FACS cellular uptake of d-arginine oligomersand polyguanidine peptoids. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

[0057]FIG. 28 displays the FACS cellular uptake of and d-arginineoligomers and N-hxg peptoids. Jurkat cells were incubated with varyingconcentrations (6.3 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

[0058]FIG. 29 shows the FACS cellular uptake of d-arginine oligomers andN-chg peptoids. Jurkat cells were incubated with varying concentrations(12.5 μM shown) of fluorescently labeled peptoids and peptides for 4 minat 23° C.

[0059]FIG. 30 shows a general strategy for attaching adelivery-enhancing transporter to a drug that includes a triazole ringstructure.

[0060]FIG. 31A and FIG. 31B show synthetic schemes for making conjugatesin which FK506 is attached to a delivery-enhancing transporter.

[0061]FIG. 32 show that short oligomers of arginine, but not lysine,effectively enter Caco-2 cells. Caco-2 cells were incubated with varyingconcentrations with each of the peptides shown, washed, and analyzed byflow cytometry. Uptake could be inhibited with preincubation with sodiumazide, demonstrating that it was energy dependent.

[0062]FIG. 33 displays the measured fluorescence of Caco-2 cells exposedto fluorescent taxol or fluorescent, nonreleasable taxol conjugates witheither heptamers (r7) or decamers (r10) of D-arginine.

[0063]FIG. 34 demonstrates Caco-2 monolayer integrity by the measurementof a stable transepithlial electric resistance of greater than 100 ohmcm² for the duration of the experiments described in this report.

[0064]FIG. 35 shows the accumulation of either Fl aca r5, Fl aca r9,Lucifer Yellow, or hydrocortisone in the basolateral chamber of adiffusion apparatus after transport through a Caco-2 cell monolayerafter one hour.

[0065]FIG. 36 displays the blood levels of CsA in rats measured by LC MSMS at thirty minute intervals after intracolonic injection. CsA wasadministered in Cremophor El: ethanol, 1:1, whereas the CsA conjugateswere administered in PBS. The half life in PBS, pH 7.4 at 37° C., of theCsA conjugate (CGC1072) was 1.5 hours.

[0066]FIG. 37 displays the blood levels of taxol in rats measured by LCMS MS at thirty minute intervals after intracolonic injection. Taxol wasadministered in Cremophor EL:ethanol, 1:1, whereas the two taxolconjugates were administered in PBS. The half life in PBS, pH 7.4 at 37°C., of the slower releasing conjugate (14) was 5 hours, while that ofthe faster conjugate (13) was ten minutes. See, Example 18.

[0067]FIG. 38 displays the blood levels of taxol in rats measured by LCMS MS at thirty minute intervals after buccal delivery. Taxol wasadministered in Cremophor El: ethanol, 1:1, whereas the taxol conjugatewas administered in PBS. The half life in PBS, pH 7.4 at 37° C., of theconjugate (13) was ten minutes.

[0068]FIG. 39 illustrates the conjugation of acyclovir to r7-amide viaan N-terminal cysteine group. Conjugation with a biotin-containingtransporter is also shown.

[0069]FIG. 40 illustrates the conjugate formed between a retinal and ar9 (shown without spacing amino acids).

[0070]FIG. 41 illustrates the use of a cleavable linker in preparing aretinoic acid-r9 conjugate.

DETAILED DESCRIPTION

[0071] Definitions

[0072] An “epithelial tissue” is the basic tissue that covers surfaceareas of the surface, spaces, and cavities of the body. Epithelialtissues are composed primarily of epithelial cells that are attached toone another and rest on an extracellular matrix (basement membrane) thatis typically produced by the cells. Epithelial tissues include threegeneral types based on cell shape: squamous, cuboidal, and columnarepithelium. Squamous epithelium, which lines lungs and blood vessels, ismade up of flat cells. Cuboidal epithelium lines kidney tubules and iscomposed of cube shaped cells, while columnar epithelium cells line thedigestive tract and have a columnar appearance. Epithelial tissues canalso be classified based on the number of cell layers in the tissue. Forexample, a simple epithelial tissue is composed of a single layer ofcells, each of which sits on the basement membrane. A “stratified”epithelial tissue is composed of several cells stacked upon one another;not all cells contact the basement membrane. A “pseudostratified”epithelial tissue has cells that, although all contact the basementmembrane, appear to be stratified because the nuclei are at variouslevels.

[0073] The term “trans-epithelial” delivery or administration refers tothe delivery or administration of agents by permeation through one ormore layers of a body surface or tissue, such as intact skin or a mucousmembrane, by topical administration. Thus, the term is intended toinclude both transdermal (e.g., percutaneous adsorption) andtransmucosal administration. Delivery can be to a deeper layer of thetissue, for example, and/or delivery to the bloodstream.

[0074] “Delivery enhancement, “penetration enhancement” or “permeationenhancement” as used herein relates to an increase in amount and/or rateof delivery of a compound that is delivered into and across one or morelayers of an epithelial or endothelial tissue. An enhancement ofdelivery can be observed by measuring the rate and/or amount of thecompound that passes through one or more layers of animal or human skinor other tissue. Delivery enhancement also can involve an increase inthe depth into the tissue to which the compound is delivered, and/or theextent of delivery to one or more cell types of the epithelial or othertissue (e.g., increased delivery to fibroblasts, immune cells, andendothelial cells of the skin or other tissue). Such measurements arereadily obtained by, for example, using a diffusion cell apparatus asdescribed in U.S. Pat. No. 5,891,462.

[0075] The amount or rate of delivery of an agent across and/or intoskin or other epithelial or endothelial membrane is sometimesquantitated in terms of the amount of compound passing through apredetermined area of skin or other tissue, which is a defined area ofintact unbroken living skin or mucosal tissue. That area will usually bein the range of about 5 cm² to about 100 cm², more usually in the rangeof about 10 cm² to about 100 cm², still more usually in the range ofabout 20 cm² to about 60 cm².

[0076] The terms “guanidyl,” guanidinyl” and “guanidino” are usedinterchangeably to refer to a moiety having the formula —HN═C(NH₂)NH(unprotonated form). As an example, arginine contains a guanidyl(guanidino) moiety, and is also referred to as2-amino-5-guanidinovaleric acid or α-amino-δ-guanidinovaleric acid.“Guanidium” refers to the positively charged conjugate acid form. Theterm “guanidino moiety” includes, for example, guanidine, guanidinium,guanidine derivatives such as (RNHC(NH)NHR′), monosubstitutedguanidines, monoguanides, biguanides, biguanide derivatives such as(RNHC(NH)NHC(NH)NHR′), and the like. In addition, the term “guanidinomoiety” encompasses any one or more of a guanide alone or a combinationof different guanides.

[0077] “Amidinyl” and “amidino” refer to a moiety having the formula—C(═NH)(NH₂). “Amidinium” refers to the positively charged conjugateacid form.

[0078] The term “trans-barrier concentration” or “trans-tissueconcentration” refers to the concentration of a compound present on theside of one or more layers of an epithelial or endothelial barriertissue that is opposite or “trans” to the side of the tissue to which aparticular composition has been added. For example, when a compound isapplied to the skin, the amount of the compound measured subsequentlyacross one or more layers of the skin is the trans-barrier concentrationof the compound.

[0079] “Biologically active agent” or “biologically active substance”refers to a chemical substance, such as a small molecule, macromolecule,or metal ion, that causes an observable change in the structure,function, or composition of a cell upon uptake by the cell. Observablechanges include increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, increased or decreased cell proliferation, and thelike.

[0080] The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccharides, for example.

[0081] The term “macromolecule” as used herein refers to large molecules(MW greater than 1000 daltons) exemplified by, but not limited to,peptides, proteins, oligonucleotides and polynucleotides of biologicalor synthetic origin.

[0082] “Small organic molecule” refers to a carbon-containing agenthaving a molecular weight (MW) of less than or equal to 1000 daltons.

[0083] The terms “non-polypeptide agent” and “non-polypeptidetherapeutic agent” refer to the portion of a conjugate that does notinclude the delivery-enhancing transporter, and that is a biologicallyactive agent other than a polypeptide. An example of a non-polypeptideagent is an anti-sense oligonucleotide, which can be conjugated to apoly-arginine peptide to form a conjugate for enhanced delivery into andacross one or more layers of an epithelial or endothelial tissue.

[0084] A “subunit,” as used herein, is a monomeric unit that are joinedto form a larger polymeric compound. The set of amino acids are anexample of subunits. Each amino acid shares a common backbone (—C—C—N—),and the different amino acids differ in their sidechains. The backboneis repeated in a polypeptide. A subunit represents the shortestrepeating pattern of elements in a polymer backbone. For example, twoamino acids of a peptide are not considered a peptide because two aminoacids would not have the shortest repeating pattern of elements in thepolymer backbone.

[0085] The term “polymer” refers to a linear chain of two or moreidentical or non-identical subunits joined by covalent bonds. A peptideis an example of a polymer; peptides can be composed of identical ornon-identical amino acid subunits that are joined by peptide linkages(amide bonds).

[0086] The term “peptide” as used herein refers to a compound made up ofa single chain of D- or L-amino acids or a mixture of D- and L-aminoacids joined by peptide bonds. Generally, peptides contain at least twoamino acid residues and are less than about 50 amino acids in length.D-amino acids are represented herein by a lower-case one-letter aminoacid symbol (e.g., r for D-arginine), whereas L-amino acids arerepresented by an upper case one-letter amino acid symbol (e.g., R forL-arginine). Homopolymer peptides are represented by a one-letter aminoacid symbol followed by the number of consecutive occurrences of thatamino acid in the peptide—(e.g., R7 represents a heptamer that consistsof L-arginine residues).

[0087] The term “protein” as used herein refers to a compound that iscomposed of linearly arranged amino acids linked by peptide bonds, butin contrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

[0088] “Polypeptide” as used herein refers to a polymer of at least twoamino acid residues and which contains one or more peptide bonds.“Polypeptide” encompasses peptides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

[0089] Description of the Preferred Embodiments

[0090] The present invention provides compositions and methods thatenhance the transfer of compounds, including drugs and otherbiologically active compounds, into and across one or more layers of ananimal epithelial or endothelial tissue. The methods involve contactingthe tissue with a conjugate that includes the compound of interestlinked to a delivery-enhancing transporter. The delivery enhancingtransporters provided by the invention are molecules that includesufficient guanidino or amidino moieties to increase delivery of theconjugate into and across one or more intact epithelial and endothelialtissue layers. The methods and compositions are useful fortrans-epithelial and trans-endothelial delivery of drugs and otherbiologically active molecules, and also for delivery of imaging anddiagnostic molecules. The methods and compositions of the invention areparticularly useful for delivery of compounds that requiretrans-epithelial or trans-endothelial transport to exhibit theirbiological effects, and that by themselves (without conjugation to adelivery-enhancing transporters or some other modification), are unable,or only poorly able, to cross such tissues and thus exhibit biologicalactivity.

[0091] The delivery-enhancing transporters and methods of the inventionprovide significant advantages over previously available methods forobtaining trans-epithelial and trans-endothelial tissue delivery ofcompounds of interest. The transporters make possible the delivery ofdrugs and other agents across tissues that were previously impenetrableto the drug. For example, while delivery of drugs across skin waspreviously nearly impossible for all but a few compounds, the methods ofthe invention can deliver compounds not only into cells of a first layerof an epithelial tissue such as skin, but also across one or more layersof the skin. The blood brain barrier is also resistant to transport ofdrugs and other diagnostic and therapeutic reagents; the methods andtransporters of the invention provide means to obtain such transport.

[0092] The delivery-enhancing transporters increase delivery of theconjugate into and across one or more intact epithelial or endothelialtissue layers compared to delivery of the compound in the absence of thedelivery-enhancing transporter. The delivery-enhancing transporters can,in some embodiments, increase delivery of the conjugate significantlyover that obtained using the tat protein of HIV-1 (Frankel et al. (1991)PCT Pub. No. WO 91/09958). Delivery is also increased significantly overthe use of shorter fragments of the tat protein containing the tat basicregion (residues 49-57 having the sequence RKKRRQRRR) (Barsoum et al.(1994) WO 94/04686 and Fawell et al. (1994) Proc. Nat'l. Acad. Sci. USA91: 664-668). Preferably, delivery obtained using the transporters ofthe invention is increased more than 2-fold, still more preferablysix-fold, still more preferably ten-fold, and still more preferablytwenty-fold, over that obtained with tat residues 49-57. In someembodiments, the compositions of the invention do not include tatresidues 49-57.

[0093] Similarly, the delivery-enhancing transporters of the inventioncan provide increased delivery compared to a 16 amino acidpeptide-cholesterol conjugate derived from the Antennapedia homeodomainthat is rapidly internalized by cultured neurons (Brugidou et al. (1995)Biochem. Biophys. Res. Commun. 214: 685-93). This region, residues 43-58at minimum, has the amino acid sequence RQIKIWFQNRRMKWKK. The Herpessimplex protein VP22, like tat and the Antennapedia domain, waspreviously known to enhance transport into cells, but was not known toenhance transport into and across endothelial and epithelial membranes(Elliot and O'Hare (1997) Cell 88: 223-33; Dilber et al. (1999) GeneTher. 6: 12-21; Phelan et al. (1998) Nature Biotechnol. 16: 440-3). Inpresently preferred embodiments, the delivery-enhancing transportersprovide significantly increased delivery compared to the Antennapediahomeodomain and to the VP22 protein. In some embodiments, thecompositions of the invention do not include the Antennapediahomeodomain, the VP22 protein or eight contiguous arginines.

[0094] Structure of Delivery-Enhancing Transporters

[0095] The delivery-enhancing transporters of the invention aremolecules that have sufficient guanidino and/or amidino moieties toincrease delivery of a compound to which the delivery-enhancingtransporter is attached into and across one or more layers of anepithelial tissue (e.g., skin or mucous membrane) or an endothelialtissue (e.g., the blood-brain barrier). The delivery-enhancingtransporters generally include a backbone structure to which is attachedthe guanidino and/or amidino sidechain moieties. In some embodiments,the backbone is a polymer that consists of subunits (e.g., repeatingmonomer units), at least some of which subunits contain a guanidino oramidino moiety.

[0096] A. Guanidino and/or Amidino Moieties

[0097] The delivery-enhancing transporters typically display at least 5guanidino and/or amidino moieties, and more preferably 7 or more suchmoieties. Preferably, the delivery-enhancing transporters have 25 orfewer guanidino and/or amidino moieties, and often have 15 or fewer ofsuch moieties. In some embodiments, the delivery-enhancing transporterconsists essentially of 50 or fewer subunits, and can consistessentially of 25 or fewer, 20 or fewer, or 15 or fewer subunits. Thedelivery-enhancing transporter can be as short as 5 subunits, in whichcase all subunits include a guanidino or amidino sidechain moiety. Thedelivery-enhancing transporters can have, for example, at least 6subunits, and in some embodiments have at least 7 or 10 subunits.Generally, at least 50% of the subunits contain a guanidino or amidinosidechain moiety. More preferably, at least 70% of the subunits, andsometimes at least 90% of the subunits in the delivery-enhancingtransporter contain a guanidino or amidino sidechain moiety.

[0098] Some or all of the guanidino and/or amidino moieties in thedelivery-enhancing transporters can be contiguous. For example, thedelivery-enhancing transporters can include from 6 to 25 contiguousguanidino and/or amidino-containing subunits. Seven or more contiguousguanidino and/or amidino-containing subunits are present in someembodiments. In some embodiments, each subunit that contains a guanidinomoiety is contiguous, as exemplified by a polymer containing at leastsix contiguous arginine residues.

[0099] The delivery-enhancing transporters are exemplified by peptides.Arginine residues or analogs of arginine can constitute the subunitsthat have a guanidino moiety. Such an arginine-containing peptide can becomposed of either all D-, all L- or mixed D- and L-amino acids, and caninclude additional amino acids, amino acid analogs, or other moleculesbetween the arginine residues. Optionally, the delivery-enhancingtransporter can also include a non-arginine residue to which a compoundto be delivered is attached, either directly or through a linker. Theuse of at least one D-arginine in the delivery-enhancing transporterscan enhance biological stability of the transporter during transit ofthe conjugate to its biological target. In some cases thedelivery-enhancing transporters are at least about 50% D-arginineresidues, and for even greater stability transporters in which all ofthe subunits are D-arginine residues are used. If the delivery enhancingtransporter molecule is a peptide, the transporter is not attached to anamino acid sequence to which the amino acids that make up the deliveryenhancing transporter molecule are attached in a naturally occurringprotein.

[0100] Preferably, the delivery-enhancing transporter is linear. In apreferred embodiment, an agent to be delivered into and across one ormore layers of an epithelial tissue is attached to a terminal end of thedelivery-enhancing transporter. In some embodiments, the agent is linkedto a single transport polymer to form a conjugate. In other embodiments,the conjugate can include more than one delivery-enhancing transporterlinked to an agent, or multiple agents linked to a singledelivery-enhancing transporter.

[0101] More generally, it is preferred that each subunit contains ahighly basic sidechain moiety which (i) has a pKa of greater than 11,more preferably 12.5 or greater, and (ii) contains, in its protonatedstate, at least two geminal amino groups (NH2) which share aresonance-stabilized positive charge, which gives the moiety a bidentatecharacter.

[0102] The guanidino or amidino moieties extend away from the backboneby virtue of being linked to the backbone by a sidechain linker. Thesidechain atoms are preferably provided as methylene carbon atoms,although one or more other atoms such as oxygen, sulfur or nitrogen canalso be present. For example, a linker that attaches a guanidino moietyto a backbone can be shown as:

[0103] In these formulae, n is preferably at least 2, and is preferablybetween 2 and 7. In some embodiments, n is 3 (arginine for structure 1).In other embodiments, n is between 4 and 6; most preferably n is 5 or 6.Although the sidechain in the exemplified formulae is shown as beingattached to a peptide backbone (i.e., a repeating amide to which thesidechain is attached to the carbon atom that is α to the carbonylgroup, subunit 1) and a peptoid backbone (i.e., a repeating amide towhich the sidechain is attached to the nitrogen atom that is β to thecarbonyl group, subunit 2), other non-peptide backbones are alsosuitable, as discussed in more detail herein. Thus, similar sidechainlinkers can be attached to nonpeptide backbones (e.g., peptoidbackbones).

[0104] In some embodiments, the delivery-enhancing transporters arecomposed of linked subunits, at least some of which include a guanidinoand/or amidino moiety. Examples of suitable subunits having guanidinoand/or amidino moieties are described below.

[0105] Amino acids.

[0106] In some embodiments, the delivery-enhancing transporters arecomposed of D or L amino acid residues. The amino acids can be naturallyoccurring or non-naturally occurring amino acids. Arginine(α-amino-δ-guanidinovaleric acid) and α-amino-ε-amidino-hexanoic acid(isosteric amidino analog) are examples of suitable guanidino- andamidino-containing amino acid subunits. The guanidinium group inarginine has a pKa of about 12.5. In some preferred embodiments thetransporters are comprised of at least six contiguous arginine residues.

[0107] Other amino acids, such as α-amino-β-guanidino-propionic acid,α-amino-γ-guanidino-butyric acid, or α-amino-ε-guanidino-caproic acid(containing 2, 3 or 5 sidechain linker atoms, respectively, between thebackbone chain and the central guanidinium carbon) can also be used.

[0108] D-amino acids can also be used in the delivery enhancingtransporters.

[0109] Compositions containing exclusively D-amino acids have theadvantage of decreased enzymatic degradation. However, they can alsoremain largely intact within the target cell. Such stability isgenerally not problematic if the agent is biologically active when thepolymer is still attached. For agents that are inactive in conjugateform, a linker that is cleavable at the site of action (e.g., by enzyme-or solvent-mediated cleavage within a cell) should be included withinthe conjugate to promote release of the agent in cells or organelles.

[0110] In addition, the transport moieties are amino acid oligomers ofthe following formulae: (ZYZ)_(n)Z, (ZY)_(n)Z, (ZYY)_(n)Z and(ZYYY)_(n)Z. See, U.S. patent application Ser. No. ______, filed Feb.16, 2001 (Attorney docket No. 019801-001000US. “Z” in the formulae is Dor L-arginine. “Y” is an amino acid that does not contain a guanidyl oramidinyl moiety. The subscript “n” is an integer ranging from 2 to 25.

[0111] In the above transport moiety formulae, the letter “Y” representsa natural or non-natural amino acid. The amino acid can be essentiallyany compound having (prior to incorporation into the transport moiety)an amino group (NH₂ or NH-alkyl) and a carboxylic acid group (CO₂H) andnot containing either a guanidyl or amidinyl moiety. Examples of suchcompounds include D and L-alanine, D and L-cysteine, D and L-asparticacid, D and L-glutamic acid, D and L-phenylalanine, glycine, D andL-histidine, D and L-isoleucine, D and L-lysine, D and L-leucine, D andL-methionine, D and L-asparagine, D and L-proline, D and L-glutamine, Dand L-serine, D and L-threonine, D and L-valine, D and L-tryptophan, Dand L-hydroxyproline, D and L-tyrosine, sarcosine, β-alanine, γ-aminobutyric acid and ε-amino caproic acid. In each of the above formulae,each Y will be independent of any other Y present in the transportmoiety, though in some embodiments, all Y groups can be the same.

[0112] In one group of preferred embodiments, the transport moiety hasthe formula (ZYZ)_(n)Z, wherein each “Y” is independently selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 2to 5. More preferably, each “Y” is glycine or ε-amino caproic acid and nis 3. Within this group of embodiments, the use of glycine is preferredfor those compositions in which the transport moiety is fused orcovalently attached directly to a polypeptide biological agent such thatthe entire composition can be prepared by recombinant methods. For thoseembodiments in which the transport moiety is to be assembled using, forexample, solid phase methods, ε-amino caproic acid is preferred.

[0113] In another group of preferred embodiments, the transport moietyhas the formula (ZY)_(n)Z, wherein each “Y” is preferably selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 4to 10. More preferably, each “Y” is glycine or ε-amino caproic acid andn is 6. As with the above group of specific embodiments, the use ofglycine is preferred for those compositions in which the transportmoiety is fused or covalently attached directly to a polypeptidebiological agent such that the entire composition can be prepared byrecombinant methods. For solution or solid phase construction of thetransport moiety, ε-amino caproic acid is preferred.

[0114] In yet another group of preferred embodiments, the transportmoiety has the formula (ZYY)_(n)Z, wherein each “Y” is preferablyselected from glycine, β-alanine, γ-amino butyric acid and ε-aminocaproic acid, “Z” is preferably L-arginine, and n is preferably aninteger ranging from 4 to 10. More preferably, each “Y” is glycine orε-amino caproic acid and n is 6.

[0115] In still another group of preferred embodiments, the transportmoiety has the formula (ZYYY)_(n)Z, wherein each “Y” is preferablyselected from glycine, β-alanine, γ-amino butyric acid and ε-aminocaproic acid, “Z” is preferably L-arginine, and n is preferably aninteger ranging from 4 to 10. More preferably, “Y” is glycine and n is6.

[0116] In other embodiments, each of the Y groups will be selected toenhance certain desired properties of the transport moeity. For example,when transport moeities having a more hydrophobic character are desired,each Y can be selected from those naturally occuring amino acids thatare typically grouped together as hydrophobic amino acids (e.g.,phenylalanine, phenylglycine, valine, leucine, isoleucine). Similarly,transport moieties having a more hydrophilic character can be preparedwhen some or all of the Y groups are hydrophilic amino acids (e.g.,lysine, serine, threonine, glutamic acid, and the like).

[0117] One of skill in the art will appreciate that the transport moietycan be a polypeptide fragment within a larger polypeptide. For example,the transport moiety can be of the formula (ZYY)_(n)Z yet haveadditional amino acids which flank this moiety (e.g.,X_(m)(ZYY)_(n)Z-X_(p) wherein the subscripts m and p represent integersof zero to about 10 and each X is independently a natural or non-naturalamino acid).

[0118] Other Subunits.

[0119] Subunits other than amino acids can also be selected for use informing transport polymers. Such subunits can include, but are notlimited to, hydroxy amino acids, N-methyl-amino acids amino aldehydes,and the like, which result in polymers with reduced peptide bonds. Othersubunit types can be used, depending on the nature of the selectedbackbone, as discussed in the next section.

[0120] B. Backbones

[0121] The guanidino and/or amidino moieties that are included in thedelivery-enhancing transporters are generally attached to a linearbackbone. The backbone can comprise a variety of atom types, includingcarbon, nitrogen, oxygen, sulfur and phosphorus, with the majority ofthe backbone chain atoms typically consisting of carbon. A plurality ofsidechain moieties that include a terminal guanidino or amidino groupare attached to the backbone. Although spacing between adjacentsidechain moieties is typically consistent, the delivery-enhancingtransporters used in the invention can also include variable spacingbetween sidechain moieties along the backbone.

[0122] A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), keto-methylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO₂RNH), retropeptide (NHCO),trans-alkene (CR═CH), fluoroalkene (CF═CH), dimethylene (CH₂CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₄), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted amides), and backbones withmalonate and/or gem-diamino-alkyl subunits, for example, as reviewed byFletcher et al. ((1998) Chem. Rev. 98:763) and detailed by referencescited therein. Many of the foregoing substitutions result inapproximately isosteric polymer backbones relative to backbones formedfrom α-amino acids.

[0123] As mentioned above, in a peptoid backbone, the sidechain isattached to the backbone nitrogen atoms rather than the carbon atoms.(See e.g., Kessler (1993) Angew. Chem. Int. Ed. Engl. 32:543; Zuckermanet al. (1992) Chemtracts-Macromol. Chem. 4:80; and Simon et al. (1992)Proc. Nat'l. Acad. Sci. USA 89:9367.) An example of a suitable peptoidbackbone is poly-(N-substituted)glycine (poly-NSG). Synthesis ofpeptoids is described in, for example, U.S. Pat. No. 5,877,278. As theterm is used herein, transporters that have a peptoid backbone areconsidered “non-peptide” transporters, because the transporters are notcomposed of amino acids having naturally occurring sidechain locations.Non-peptide backbones, including peptoid backbones, provide enhancedbiological stability (for example, resistance to enzymatic degradationin vivo).

[0124] C. Synthesis of Delivery-Enhancing Transporters

[0125] Delivery-enhancing transporters are constructed by any methodknown in the art. Exemplary peptide polymers can be producedsynthetically, preferably using a peptide synthesizer (e.g., an AppliedBiosystems Model 433) or can be synthesized recombinantly by methodswell known in the art. Recombinant synthesis is generally used when thedelivery enhancing transporter is a peptide which is fused to apolypeptide or protein of interest.

[0126] N-methyl and hydroxy-amino acids can be substituted forconventional amino acids in solid phase peptide synthesis. However,production of delivery-enhancing transporters with reduced peptide bondsrequires synthesis of the dimer of amino acids containing the reducedpeptide bond. Such dimers are incorporated into polymers using standardsolid phase synthesis procedures. Other synthesis procedures are wellknown and can be found, for example, in Fletcher et al. (1998) Chem.Rev. 98:763, Simon et al. (1992) Proc. Nat'l. Acad. Sci. USA 89:9367,and references cited therein.

[0127] The delivery-enhancing transporters of the invention can beflanked by one or more non-guanidino/non-amidino subunits (such asglycine, alanine, and cysteine, for example), or a linker (such as anaminocaproic acid group), that do not significantly affect the rate oftrans-tissue layer transport of the corresponding delivery-enhancingtransporter-containing conjugates. Also, any free amino terminal groupcan be capped with a blocking group, such as an acetyl or benzyl group,to prevent ubiquitination in vivo.

[0128] Where the transporter is a peptoid polymer, one synthetic methodinvolves the following steps: 1) a peptoid polyamine is treated with abase and pyrazole-1-carboxamidine to provide a mixture; 2) the mixtureis heated and then allowed to cool; 3) the cooled mixture is acidified;and 4) the acidified mixture is purified. Preferably the base used instep 1 is a carbonate, such as sodium carbonate, and heating step 2involves heating the mixture to approximately 50° C. for between about24 hours and about 48 hours. The purification step preferably involveschromatography (e.g., reverse-phase HPLC).

[0129] D. Attachment of Transport Polymers to Biologically Active Agents

[0130] The agent to be transported can be linked to thedelivery-enhancing transporter according to a number of embodiments. Inone embodiment, the agent is linked to a single delivery-enhancingtransporter, either via linkage to a terminal end of thedelivery-enhancing transporter or to an internal subunit within thereagent via a suitable linking group.

[0131] In a second embodiment, the agent is attached to more than onedelivery-enhancing transporter, in the same manner as above. Thisembodiment is somewhat less preferred, since it can lead to crosslinkingof adjacent cells.

[0132] In a third embodiment, the conjugate contains two agent moietiesattached to each terminal end of the delivery-enhancing transporter. Forthis embodiment, it is presently preferred that the agent has amolecular weight of less than 10 kDa.

[0133] With regard to the first and third embodiments just mentioned,the agent is generally not attached to one any of the guanidino oramidino sidechains so that they are free to interact with the targetmembrane.

[0134] The conjugates of the invention can be prepared bystraightforward synthetic schemes. Furthermore, the conjugate productsare usually substantially homogeneous in length and composition, so thatthey provide greater consistency and reproducibility in their effectsthan heterogeneous mixtures.

[0135] According to an important aspect of the present invention, it hasbeen found by the applicants that attachment of a singledelivery-enhancing transporter to any of a variety of types ofbiologically active agents is sufficient to substantially enhance therate of uptake of an agent into and across one or more layers ofepithelial and endothelial tissues, even without requiring the presenceof a large hydrophobic moiety in the conjugate. In fact, attaching alarge hydrophobic moiety can significantly impede or prevent cross-layertransport due to adhesion of the hydrophobic moiety to the lipid bilayerof cells that make up the epithelial or endothelial tissue. Accordingly,the present invention includes conjugates that do not containsubstantially hydrophobic moieties, such as lipid and fatty acidmolecules.

[0136] Delivery-enhancing transporters of the invention can be attachedcovalently to biologically active agents by chemical or recombinantmethods.

[0137] 1. Chemical Linkages

[0138] Biologically active agents such as small organic molecules andmacromolecules can be linked to delivery-enhancing transporters of theinvention via a number of methods known in the art (see, for example,Wong, S. S., Ed., Chemistry of Protein Conjugation and Cross-Linking,CRC Press, Inc., Boca Raton, Fla. (1991), either directly (e.g., with acarbodiimide) or via a linking moiety. In particular, carbamate, ester,thioether, disulfide, and hydrazone linkages are generally easy to formand suitable for most applications. Ester and disulfide linkages arepreferred if the linkage is to be readily degraded in the cytosol, aftertransport of the substance across the cell membrane.

[0139] Various functional groups (hydroxyl, amino, halogen, etc.) can beused to attach the biologically active agent to the transport polymer.Groups which are not known to be part of an active site of thebiologically active agent are preferred, particularly if the polypeptideor any portion thereof is to remain attached to the substance afterdelivery.

[0140] Polymers, such as peptides produced as described in PCTapplication US98/10571 (Publication No. WO 9852614), are generallyproduced with an amino terminal protecting group, such as FMOC. Forbiologically active agents which can survive the conditions used tocleave the polypeptide from the synthesis resin and deprotect thesidechains, the FMOC may be cleaved from the N-terminus of the completedresin-bound polypeptide so that the agent can be linked to the freeN-terminal amine. In such cases, the agent to be attached is typicallyactivated by methods well known in the art to produce an active ester oractive carbonate moiety effective to form an amide or carbamate linkage,respectively, with the polymer amino group. Of course, other linkingchemistries can also be used.

[0141] To help minimize side-reactions, guanidino and amidino moietiescan be blocked using conventional protecting groups, such ascarbobenzyloxy groups (CBZ), di-t-BOC, PMC, Pbf, N—NO₂, and the like.

[0142] Coupling reactions are performed by known coupling methods in anyof an array of solvents, such as N,N-dimethyl formamide (DMF), N-methylpyrrolidinone, dichloromethane, water, and the like. Exemplary couplingreagents include, for example, O-benzotriazolyloxy tetramethyluroniumhexafluorophosphate (HATU), dicyclohexyl carbodiimide,bromo-tris(pyrrolidino) phosphonium bromide (PyBroP), etc. Otherreagents can be included, such as N,N-dimethylamino pyridine (DMAP),4-pyrrolidino pyridine, N-hydroxy succinimide, N-hydroxy benzotriazole,and the like.

[0143] 2. Fusion Polypeptides

[0144] Delivery-enhancing transporters of the invention can be attachedto biologically active polypeptide agents by recombinant means byconstructing vectors for fusion proteins comprising the polypeptide ofinterest and the delivery-enhancing transporter, according to methodswell known in the art. Generally, the delivery-enhancing transportercomponent will be attached at the C-terminus or N-terminus of thepolypeptide of interest, optionally via a short peptide linker.

[0145] 3. Releasable Linkers

[0146] The biologically active agents are, in presently preferredembodiments, attached to the delivery-enhancing transporter using alinkage that is specifically cleavable or releasable. The use of suchlinkages is particularly important for biologically active agents thatare inactive until the attached delivery-enhancing transporter isreleased. In some cases, such conjugates that consist of a drug moleculethat is attached to a delivery-enhancing transporter can be referred toas prodrugs, in that the release of the delivery-enhancing transporterfrom the drug results in conversion of the drug from an inactive to anactive form. As used herein, “cleaved” or “cleavage” of a conjugate orlinker refers to release of a biological agent from a transportermolecule, thereby releasing an active biological agent. “Specificallycleavable” or “specifically releasable” refers to the linkage betweenthe transporter and the agent being cleaved, rather than the transporterbeing degraded (e.g., by proteolytic degradation).

[0147] In some embodiments, the linkage is a readily cleavable linkage,meaning that it is susceptible to cleavage under conditions found invivo. Thus, upon passing into and through one or more layers of anepithelial and/or endothelial tissue, the agent is released from thedelivery-enhancing transporter. Readily cleavable linkages can be, forexample, linkages that are cleaved by an enzyme having a specificactivity (e.g., an esterase, protease, phosphatase, peptidase, and thelike) or by hydrolysis. For this purpose, linkers containing carboxylicacid esters and disulfide bonds are sometimes preferred, where theformer groups are hydrolyzed enzymatically or chemically, and the latterare severed by disulfide exchange, e.g., in the presence of glutathione.The linkage can be selected so it is cleavable by an enzymatic activitythat is known to be present in one or more layers of an epithelial orendothelial tissue. For example, the stratum granulosum of skin has arelatively high concentration of N-peptidase activity.

[0148] A specifically cleavable linker can be engineered onto atransporter molecule. For example, amino acids that constitute aprotease recognition site, or other such specifically recognizedenzymatic cleavage site, can be used to link the transporter to theagent. Alternatively, chemical or other types of linkers that arecleavable by, for example, exposure to light or other stimulus can beused to link the transporter to the agent of interest.

[0149] A conjugate in which an agent to be delivered and adelivery-enhancing transporter are linked by a specifically cleavable orspecifically releasable linker will have a half-life. The term“half-life” in this context refers to the amount of time required afterapplying the conjugate to an epithelial or endothelial membrane for onehalf of the amount of conjugate to become dissociated to release thefree agent. The half-life for some embodiments is typically between 5minutes and 24 hours, and more preferably is between 30 minutes and 2hours. The half-life of a conjugate can be “tuned” or modified,according to the invention, as described below.

[0150] In some embodiments, the cleavage rate of the linkers is pHdependent. For example, a linker can form a stable linkage between anagent and a delivery-enhancing transporter at an acidic pH (e.g., pH 6.5or less, more preferably about 6 or less, and still more preferablyabout 5.5 or less). However, when the conjugate is placed atphysiological pH (e.g., pH 7 or greater, preferably about pH 7.4), thelinker will undergo cleavage to release the agent. Such pH sensitivitycan be obtained by, for example, including a functional group that, whenprotonated (i.e., at an acidic pH), does not act as a nucleophile. At ahigher (e.g., physiological) pH, the functional group is no longerprotonated and thus can act as a nucleophile. Examples of suitablefunctional groups include, for example, N and S. One can use suchfunctional groups to fine-tune the pH at which self-cleavage occurs.

[0151] In another embodiment, the linking moiety is cleaved throughself-immolation. Such linking moieties in a transportmoiety-biologically active compound conjugate contain a nucleophile(e.g., oxygen, nitrogen and sulfur) distal to the biologically activecompound and a cleavable group (e.g., ester, carbonate, carbamate andthiocarbamate) proximal to the biologically active compound.Intramolecular attack of the nucleophile on the cleavable group resultsin the scission of a covalent bond, thereby releasing the linking moietyfrom the biologically active compound.

[0152] Examples of conjugates containing self-immolating linkingmoieties (e.g., biologically active agent-L-transport moiety conjugates)are represented by structures 3, 4 and 5:

[0153] wherein: R¹ is the biologically active compound; X is a linkageformed between a functional group on the biologically active compoundand a terminal functional group on the linking moiety; Y is a linkageformed from a functional group on the transport moiety and a functionalgroup on the linking moiety; A is N or CH; R² is hydrogen, alkyl, aryl,arylalkyl, acyl or allyl; R³ is the transport moiety; R⁴ is S, O, NR orCR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; R⁷ and R⁸ are independently hydrogen or alkyl; k and m areindependently either 1 or 2; and n is an integer ranging from 1 to 10.Non-limiting examples of the X and Y linkages are (in eitherorientation): —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate and phosphinate. Oneof skill in the art will appreciate that when the biological agent has ahydroxy functional group, then X will preferably be —OC(O)— or—OC(O)NH—. Similarly, when the linking group is attached to an aminoterminus of the transport moiety, Y will preferably be —C(O)NH—,—NHC(O)NH—, —SO₂NH—, —SONH— or —OC(O)NH— and the like. In each of thegroups provided above, NH is shown for brevity, but each of the linkages(X and Y) can contain substituted (e.g., N-alkyl or N-acyl) linkages aswell.

[0154] Turning first to linking groups illustrated by structure 3, anexample and preferred embodiment is illustrated for formula 3a:

[0155] wherein the wavy lines indicate points of attachment to thetransport moiety and to the biologically active compound. Preparation ofa conjugate containing this linking group is illustrated in Example 20(FIG. 6). In this Example and FIG. 6, cyclosporin A is treated withchloroacetic anhydride to form the chloroacetate ester 6i (numbering inFIG. 6) which is then combined with benzylamine to form the N-benzylglycine conjugate 6ii. Condensation of the glycine conjugate withBoc-protected diglycine anhydride provides the acid 6iii which isconverted to the more reactive N-hydroxy succinimide ester 6iv and thencombined with the amino terminus of a transport moiety to form an amidelinkage. One of skill in the art will appreciate that the N-benzyl groupcan be replaced with other groups (e.g., alkyl, aryl, allyl and thelike) or that methylene groups can be replaced with, for example,ethylene, propylene and the like. Preferably, the methylene groups areretained as shown in 3a, to provide an appropriate steric or spatialorientation that allows the linkage to be cleaved in vivo (see FIG. 6B).

[0156] Accordingly, for structure 3, the following substituents arepreferred: A is N; R² is benzyl; k, m and n are 1; X is —OC(O)— and Y is—C(O)NH—.

[0157] Linkages of structure 4, are exemplified by formula 4a:

[0158] wherein, as above, the wavy lines indicate the point ofattachment to each of the transport moiety and the biologically activeagent. The preparation of conjugates having linking groups of formula 4aare shown in Examples 20-22. In Example 20 (see scheme in FIG. 39),acyclovir is acylated with α-chloroacetic anhydride to form theα-chloroacetate ester 39i. Reaction of 39i with a heptamer of D-argininehaving an N-terminal cysteine residue, provides the thioether product39ii. Alternatively, acyclovir can be attached to the C-terminus of atransport moiety using a similar linkage formed between acyclovirα-chloroacetate ester and a heptamer of D-arginine having an C-terminalcysteine residue. In this instance, the cysteine residue is provided onthe r₇ transport moiety as a C-terminal amide and the linkage has theform:

[0159] Accordingly, in one group of preferred embodiments, the conjugateis represented by formula 5, in which X is —OC(O)—; Y is —C(O)NH—; R⁴ isS; R⁵ is NHR⁶; and the subscripts k and m are each 1. In another groupof preferred embodiments, the conjugate is represented by formula 2, inwhich X is —OC(O)—; Y is —NHC(O)—; R⁴ is S; R⁵ is CONH₂; and thesubscripts k and m are each 1. Particularly preferred conjugates arethose in which R⁶ is hydrogen, methyl, allyl, butyl or phenyl.

[0160] Linking groups represented by the conjugates shown in formula 6are generally of the heterobifunctional type (e.g., ε-aminocaproic acid,serine, homoserine, γ-aminobutyric acid, and the like), althoughsuitably protected dicarboxylic acids or diamines are also useful withcertain biological agents.

[0161] For structure 6, the following substituents are preferred: R⁵ isNHR⁶, wherein R⁶ is hydrogen, methyl, allyl, butyl or phenyl; k is 2; Xis —C(O)O—; and Y is —C(O)NH—.

[0162] Self-immolating linkers typically undergo intramolecular cleavagewith a half-life between about 10 minutes and about 24 hours in water at37° C. at a pH of approximately 7.4. Preferably, the cleavage half-lifeis between about 20 minutes and about 4 hours in water at 37° C. at a pHof approximately 7.4. More preferably, the cleavage half-life is betweenabout 30 minutes and about 2 hours in water at 37° C. at a pH ofapproximately 7.4.

[0163] For a conjugate having the structure 3, one can adjust thecleavage half-life by varying the R² substituent. By using an R² ofincreased or decreased size, one can obtain a conjugate having a longeror shorter half-life respectively. R² in structure 3 is preferablymethyl, ethyl, propyl, butyl, allyl, benzyl or phenyl.

[0164] Where there is a basic or acidic group in a self-immolatinglinker, one can oftentimes adjust cleavage half-life according to the pHof the conjugate solution. For instance, the backbone amine group ofstructure 3 is protonated at acidic pH (e.g., pH 5.5). The amine cannotserve as a nucleophile inducing intramolecular cleavage when it isprotonated. Upon introduction of the conjugate into a medium atphysiological pH (7.4), however, the amine is unprotonated a significantportion of the time. The cleavage half-life is correspondingly reduced.

[0165] In one embodiment, cleavage of a self-immolating linker occurs intwo steps: intramolecular reaction of a nucleophilic group resulting inthe cleavage of a portion of the linking moiety; and, elimination of theremaining portion of the linking moiety. The first step of the cleavageis rate-limiting and can be fine-tuned for pH sensitivity and half-life.

[0166] Structure 6 is an example of a two-step, self-immolating moietythat is incorporated into a transport moiety-biologically activecompound conjugate:

[0167] wherein: R¹ is the biologically active compound; X represents alinkage between a functional group on the biologically active compoundand a functional group on the linking moiety; Ar is a substituted orunsubstituted aryl group, wherein the methylene substituent and phenolicoxygen atom are either ortho or para to one another; R³ is the transportmoiety; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently hydrogen or alkyl; and, k and m are independently either 1or 2.

[0168] An example of a suitable linking group to produce a conjugate offormula 6 is:

[0169] The construction of a conjugate containing a linking group offormula 6a is provided in Example 24 (see also FIG. 14). In this example(and Figure), the α-chloroacetate ester of2,4-dimethyl-4-hydroxymethylphenol (41i) is coupled to retinoic acid(41ii) using dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine(DMAP) to provide the intermediate 41iii. Subsequent coupling of 41iiiwith a cysteine residue present on the N-terminus of an arginineheptamer transport moiety provides the target conjugate 41iv.

[0170] Preferably, the linking groups used in the conjugates of formula6, are those in which Ar is an substituted or unsubstituted phenylenegroup; R⁴ is S; R⁵ is NHR⁶, wherein R⁶ is hydrogen, methyl, allyl,butyl, acetyl or phenyl; k and m are 1; X is —C(O)O—; and Y is —C(O)O—or —C(O)NH—. More preferably, R⁶ is hydrogen or acetyl.

[0171] While linking groups above have been described with reference toconjugates containing arginine heptamers, one of skill in the art willunderstand that the technology is readily adapted to conjugates with the“spaced” arginine transport moieties of the present invention.

[0172] Still other useful linking groups for use in the presentinvention have been described in copending PCT applications. See, forexample PCT applications US98/10571 (Publication No. WO 9852614) andUS00/23440 (Publication No. ______) which describe linking groups forsimilar compositions, e.g., conjugates of biologically active agents andtransport oligomers. The linking technology described therein can beused in the present compositions in a similar manner.

[0173] Thus, in one group of embodiments, the linking moiety contains afirst cleavable group distal to the biologically active compound and asecond cleavable group proximal to the biologically active compound.Cleavage of the first cleavable group yields a nucleophile capable ofreacting intramolecularly with the second cleavable group, therebycleaving the linking moiety from the biologically active compound.Examples of methods by which the first group is cleaved includephoto-illumination and enzyme mediated hydrolysis. This methodology hasbeen illustrated for various related small molecule conjugates discussedin PCT application US98/10571 (Publication No. WO 9852614).

[0174] In one approach, the conjugate can include a disulfide linkage,as illustrated in FIG. 5A of PCT application US00/23440 (Publication No.______), (see also, PCT application US98/10571 (Publication No. WO9852614)), which shows a conjugate (I) containing a transport polymer Twhich is linked to a cytotoxic agent, 6-mercaptopurine, by anN-acetyl-protected cysteine group which serves as a linker. Thus, thecytotoxic agent is attached by a disulfide bond to the 6-mercapto group,and the transport polymer is bound to the cysteine carbonyl moiety viaan amide linkage. Cleavage of the disulfide bond by reduction ordisulfide exchange results in release of the free cytotoxic agent. Amethod for synthesizing a disulfide-containing conjugate is provided inExample 9A of PCT application US98/10571. The product described thereincontains a heptamer of Arg residues which is linked to 6-mercaptopurineby an N-acetyl-Cys-Ala-Ala linker, where the Ala residues are includedas an additional spacer to render the disulfide more accessible tothiols and reducing agents for cleavage within a cell. The linker inthis example also illustrates the use of amide bonds, which can becleaved enzymatically within a cell.

[0175] In another approach, the conjugate includes a photocleavablelinker that is cleaved upon exposure to electromagnetic radiation.Application of this methodology is provided for a related system in FIG.5B of PCT application US00/23440 (Publication No. ______) which shows aconjugate (II) containing a transport polymer T which is linked to6-mercaptopurine via a meta-nitrobenzoate linking moiety. Polymer T islinked to the nitrobenzoate moiety by an amide linkage to the benzoatecarbonyl group, and the cytotoxic agent is bound via its 6-mercaptogroup to the p-methylene group. The compound can be formed by reacting6-mercaptopurine with p-bromomethyl-m-nitrobenzoic acid in the presenceof NaOCH₃/methanol with heating, followed by coupling of the benzoatecarboxylic acid to a transport polymer, such as the amino group of aγ-aminobutyric acid linker attached to the polymer (see also, e.g.,Example 9B of PCT application US98/10571). Photo-illumination of theconjugate causes release of the 6-mercaptopurine by virtue of the nitrogroup that is ortho to the mercaptomethyl moiety. This approach findsutility in phototherapy methods as are known in the art, particularlyfor localizing drug activation to a selected area of the body.

[0176] In one group of preferred embodiments, the cleavable linkercontains first and second cleavable groups that can cooperate to cleavethe oligomer from the biologically active agent, as illustrated by thefollowing approaches. That is, the cleavable linker contains a firstcleavable group that is distal to the agent, and a second cleavablegroup that is proximal to the agent, such that cleavage of the firstcleavable group yields a linker-agent conjugate containing anucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andoligomer.

[0177] Reference is again made to co-owned and copending PCT applicationUS00/23440 (Publication No. ______), in which FIG. 5C shows a conjugate(III) containing a transport polymer T linked to the anticancer agent,5-fluorouracil (5FU). In that figure, the linkage is provided by amodified lysyl residue. The transport polymer is linked to the α-aminogroup, and the 5-fluorouracil is linked via the α-carbonyl. The lysylε-amino group has been modified to a carbamate ester of o-hydroxymethylnitrobenzene, which comprises a first, photolabile cleavable group inthe conjugate. Photo-illumination severs the nitrobenzene moiety fromthe conjugate, leaving a carbamate that also rapidly decomposes to givethe free α-amino group, an effective nucleophile. Intramolecularreaction of the α-amino group with the amide linkage to the5-fluorouracil group leads to cyclization with release of the5-fluorouracil group.

[0178] Still other linkers useful in the present invention are providedin PCT application US00/23440 (Publication No. ______). In particular,FIG. 5D of US00/23440 illustrates a conjugate (IV) containing adelivery-enhancing transporter T linked to 2′-oxygen of the anticanceragent, paclitaxel. The linkage is provided by a linking moiety thatincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.Enzymatic cleavage of the phosphate group from the conjugate affords afree phenol hydroxyl group. This nucleophilic group then reactsintramolecularly with the carboxylate ester to release free paclitaxel,fully capable of binding to its biological target. Example 9C of PCTapplication US98/10571 describes a synthetic protocol for preparing thistype of conjugate.

[0179] Still other suitable linkers are illustrated in FIG. 5E of PCTapplication US00/23440 (Publication No. ______). In the approachprovided therein, a delivery-enhancing transporter is linked to abiologically active agent, e.g., paclitaxel, by an aminoalkyl carboxylicacid. Preferably, the linker amino group is linked to the linkercarboxyl carbon by from 3 to 5 chain atoms (n=3 to 5), preferably either3 or 4 chain atoms, which are preferably provided as methylene carbons.As seen in FIG. 5E, the linker amino group is joined to thedelivery-enhancing transporter by an amide linkage, and is joined to thepaclitaxel moiety by an ester linkage. Enzymatic cleavage of the amidelinkage releases the delivery-enhancing transporter and produces a freenucleophilic amino group. The free amino group can then reactintramolecularly with the ester group to release the linker from thepaclitaxel.

[0180] In another approach, the conjugate includes a linker that islabile at one pH but is stable at another pH. For example, FIG. 6 of PCTapplication US00/23440 (Publication No. ______) illustrates a method ofsynthesizing a conjugate with a linker that is cleaved at physiologicalpH but is stable at acidic pH. Preferably, the linker is cleaved inwater at a pH of from about 6.6 to about 7.6. Preferably the linker isstable in water at a pH from about 4.5 to about 6.5.

[0181] Uses of Delivery-Enhancing Transporters

[0182] The delivery-enhancing transporters find use in therapeutic,prophylactic and diagnostic applications. The delivery-enhancingtransporters can carry a diagnostic or biologically active reagent intoand across one or more layers of skin or other epithelial tissue (e.g.,gastrointestinal, lung, ocular and the like), or across endothelialtissues such as the blood brain barrier. This property makes thereagents useful for treating conditions by delivering agents that mustpenetrate across one or more tissue layers in order to exert theirbiological effect.

[0183] Moreover, the transporters of the present invention can also beused alone, or in combination with another therapeutic or othercompound, as a furin inhibitor. For example, in addition to variouspoly-arginine transporters, the synthetic transporters described herein,including peptoid and those transporters comprising non-naturallyoccurring amino acids can be used to inhibit furins. See, e.g., Cameronet al., J. Biol. Chem. 275(47): Furins are proteases that convert avariety of pro-proteins to their active components. Inhibition of furinsis useful, for instance, for treating infections by viruses that rely onfurin activity for virulence or replication. See, e.g., Molloy, et al.,T. Cell Biol. 9:28-35 (1999).

[0184] Similarly, the transporters of the invention are usefulinhibitors of capthesin C. For example, certain poly arginine compoundsare inhibitors of capthesin C. See, e.g., Horn, et al., Eur. J. Biochem.267(11):3330-3336 (2000). Similarly, the transporters of the invention,including those comprising synthetic amino acids, are useful to inhibitcapthesin C.

[0185] In one aspect of the invention, a furin inhibition assay can beused to screen for additional transporters. For example, candidatetransporter compounds can be tested for their ability to compete withpoly arginine for their ability to bind furins or capthesin C usingstandard competition assays. Alternatively, candidate transporters canbe screened for their ability to inhibit furin protease activity asdiscussed in Cameron et al., supra. and Horn et al., supra. Particularlyactive candidates can then be further tested for their ability to act astransporters into and/or across tissues such as the epithelium.

[0186] Compositions and methods of the present invention have particularutility in the area of human and veterinary therapeutics. Generally,administered dosages will be effective to deliver picomolar tomicromolar concentrations of the therapeutic composition to the effectorsite. Appropriate dosages and concentrations will depend on factors suchas the therapeutic composition or drug, the site of intended delivery,and the route of administration, all of which can be derived empiricallyaccording to methods well known in the art. Further guidance can beobtained from studies using experimental animal models for evaluatingdosage, as are known in the art.

[0187] Administration of the compounds of the invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,intramuscular, oral, intra-joint, parenteral, peritoneal, intranasal, orby inhalation. Suitable sites of administration thus include, but arenot limited to, skin, bronchial, gastrointestinal, anal, vaginal, eye,and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, aerosols,eye drops, or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages.

[0188] The compositions typically include a conventional pharmaceuticalcarrier or excipient and may additionally include other medicinalagents, carriers, adjuvants, and the like. Preferably, the compositionwill be about 5% to 75% by weight of a compound or compounds of theinvention, with the remainder consisting of suitable pharmaceuticalexcipients. Appropriate excipients can be tailored to the particularcomposition and route of administration by methods well known in theart, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., MackPublishing Co., Easton, Pa. (1990).

[0189] For oral administration, such excipients include pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like. The composition may take the form of asolution, suspension, tablet, pill, capsule, powder, sustained-releaseformulation, and the like.

[0190] In some embodiments, the pharmaceutical compositions take theform of a pill, tablet or capsule, and thus, the composition cancontain, along with the biologically active conjugate, any of thefollowing: a diluent such as lactose, sucrose, dicalcium phosphate, andthe like; a disintegrant such as starch or derivatives thereof; alubricant such as magnesium stearate and the like; and a binder such astarch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose andderivatives thereof.

[0191] The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

[0192] Liquid compositions can be prepared by dissolving or dispersingcompound (about 0.5% to about 20%), and optional pharmaceuticaladjuvants in a carrier, such as, for example, aqueous saline (e.g., 0.9%w/v sodium chloride), aqueous dextrose, glycerol, ethanol and the like,to form a solution or suspension, e.g., for intravenous administration.The active compounds may also be formulated into a retention enema.

[0193] If desired, the composition to be administered may also containminor amounts of non-toxic auxiliary substances such as wetting oremulsifying agents, pH buffering agents, such as, for example, sodiumacetate, sorbitan monolaurate, or triethanolamine oleate.

[0194] For topical administration, the composition is administered inany suitable format, such as a lotion or a transdermal patch. Fordelivery by inhalation, the composition can be delivered as a dry powder(e.g., Inhale Therapeutics) or in liquid form via a nebulizer.

[0195] Methods for preparing such dosage forms are known or will beapparent to those skilled in the art; for example, see Remington'sPharmaceutical Sciences, supra., and similar publications. Thecomposition to be administered will, in any event, contain a quantity ofthe pro-drug and/or active compound(s) in a pharmaceutically effectiveamount for relief of the condition being treated when administered inaccordance with the teachings of this invention.

[0196] Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

[0197] Stability of the conjugate can be further controlled by thecomposition and stereochemistry of the backbone and sidechains of thedelivery-enhancing transporters. For polypeptide delivery-enhancingtransporters, D-isomers are generally resistant to endogenous proteases,and therefore have longer half-lives in serum and within cells.D-polypeptide polymers are therefore appropriate when longer duration ofaction is desired. L-polypeptide polymers have shorter half-lives due totheir susceptibility to proteases, and are therefore chosen to impartshorter acting effects. This allows side-effects to be averted morereadily by withdrawing therapy as soon as side-effects are observed.Polypeptides comprising mixtures of D and L-residues have intermediatestabilities. Homo-D-polymers are generally preferred.

[0198] A. Application to Skin

[0199] The delivery-enhancing transporters of the invention makepossible the delivery of biologically active and diagnostic agentsacross the skin. Surprisingly, the transporters can deliver an agentacross the stratum corneum, which previously had been a nearlyimpenetrable barrier to drug delivery. The stratum corneum, theoutermost layer of the skin, is composed of several layers of dead,keratin-filled skin cells that are tightly bound together by a “glue”composed of cholesterol and fatty acids. Once the agents are deliveredthrough the stratum corneum by the transporters of the invention, theagents can enter the viable epidermis, which is composed of the stratumgranulosum, stratum lucidum and stratum germinativum which, along withthe stratum corneum, make up the epidermis. Delivery in some embodimentsof the invention is through the epidermis and into the dermis, includingone or both of the papillary dermis and the reticular dermis.

[0200] This ability to obtain penetration of one or more layers of theskin can greatly enhance the efficacy of compounds such asantibacterials, antifungals, antivirals, antiproliferatives,immunosuppressives, vitamins, analgesics, hormones, and the like.Numerous such compounds are known to those of skill in the art (see,e.g., Hardman and Limbird, Goodman & Gilman 's The Pharmacological Basisof Therapeutics, McGraw-Hill, New York, 1996).

[0201] In some embodiments, the agent is delivered into a blood vesselthat is present in the epithelial tissue, thus providing a means fordelivery of the agent systemically. Delivery can be eitherintrafollicular or interfollicular, or both. Pretreatment of the skin isnot required for delivery of the conjugates.

[0202] In other embodiments, the delivery-enhancing transporters areuseful for delivering cosmetics and agents that can treat skinconditions. Target cells in the skin that are of interest include, forexample, fibroblasts, epithelial cells and immune cells. For example,the transporters provide the ability to deliver compounds such asantiinflammatory agents to immune cells found in the dermis.

[0203] Glucocorticoids (adrenocorticoid steroids) are among thecompounds for which delivery across skin can be enhanced by thedelivery-enhancing transporters of the invention. Conjugatedglucocorticoids of the invention are useful for treating inflammatoryskin diseases, for example. Exemplary glucocorticoids include, e.g.,hydrocortisone, prenisone (deltasone) and predrisonlone (hydeltasol).Examples of particular conditions include eczema (including atopicdermatitis, contact dermatitis, allergic dermatitis), bullous disease,collagen vascular diseases, sarcoidosis, Sweet's disease, pyodermagangrenosum, Type I reactive leprosy, capillary hemangiomas, lichenplanus, exfoliative dermatitis, erythema nodosum, hormonal abnormalities(including acne and hirsutism), as well as toxic epidermal necrolysis,erythema multiforme, cutaneous T-cell lymphoma, discoid lupuserythematosus, and the like.

[0204] Retinoids are another example of a biologically active agent forwhich one can use the delivery-enhancing transporters of the inventionto enhance delivery into and across one or more layers of the skin orother epithelial or endothelial tissue. Retinoids that are presently inuse include, for example retinol, tretinoin, isotretinoin, etretinate,acitretin, and arotinoid. Conditions that are treatable using retinoidsconjugated to the delivery-enhancing transporters of the inventioninclude, but are not limited to, acne, keratinization disorders, skincancer, precancerous conditions, psoriasis, cutaneous aging, discoidlupus erythematosus, scleromyxedema, verrucous epidermal nevus,subcomeal pustular dermatosis, Reiter's syndrome, warts, lichen planus,acanthosis nigricans, sarcoidosis, Grover's disease, porokeratosis, andthe like.

[0205] Cytotoxic and immunosuppressive drugs constitute an additionalclass of drugs for which the delivery-enhancing transporters of theinvention are useful. These agents are commonly used to treathyperproliferative diseases such as psoriasis, as well as for immunediseases such as bullous dermatoses and leukocytoclastic vasculitis.Examples of such compounds that one can conjugate to thedelivery-enhancing transporters of the invention include, but are notlimited to, antimetabolites such as methotrexate, azathioprine,fluorouracil, hydroxyurea, 6-thioquanine, mycophenolate, chlorambucil,vinicristine, vinblasrine and dactinomycin. Other examples arealkylating agents such as cyclophosphamide, mechloroethaminehydrochloride, carmustine. taxol, tacrolimus and vinblastine areadditional examples of useful biological agents, as are dapsone andsulfasalazine. Immunosuppressive drugs such as cyclosporin andAscomycins, such as FK506 (tacrolimus), and rapamycin (e.g., U.S. Pat.No. 5,912,253) and analogs of such compounds are of particular interest(e.g., Mollinson et al., Current Pharm. Design 4(5):367-380 (1998); U.S.Pat. Nos. 5,612,350; 5,599,927; 5,604,294; 5,990,131; 5,561,140;5,859,031; 5,925,649; 5,994,299; 6,004,973 and 5,508,397). Cyclosporinsinclude cyclosporin A, B, C, D, G and M. See, e.g., U.S. Pat. Nos.6,007,840; and 6,004,973. For example, such compounds are useful intreating psoriasis, eczema (including atopic dermatitis, contactdermatitis, allergic dermatitis) and alopecia areata.

[0206] The delivery-enhancing transporters can be conjugated to agentsthat are useful for treating conditions such as lupus erythematosus(both discoid and systemic), cutaneous dermatomyositis, porphyriacutanea tarda and polymorphous light eruption. Agents useful fortreating such conditions include, for example, quinine, chloroquine,hydroxychloroquine, and quinacrine.

[0207] The delivery-enhancing transporters of the invention are alsouseful for transdermal delivery of antiinfective agents. For example,antibacterial, antifungal and antiviral agents can be conjugated to thedelivery-enhancing transporters. Antibacterial agents are useful fortreating conditions such as acne, cutaneous infections, and the like.Antifungal agents can be used to treat tinea corporis, tinea pedis,onychomycosis, candidiasis, tinea versicolor, and the like. Because ofthe delivery-enhancing properties of the conjugates, these conjugatesare useful for treating both localized and widespread infections.Antifungal agents are also useful for treating onychomycosis. Examplesof antifungal agents include, but are not limited to, azole antifungalssuch as itraconazole, myconazole and fluconazole. Examples of antiviralagents include, but are not limited to, acyclovir, famciclovir, andvalacyclovir. Such agents are useful for treating viral diseases, e.g.,herpes.

[0208] Another example of a biologically active agent for whichenhancement of delivery by conjugation to the delivery-enhancingtransporters of the invention is desirable are the antihistamines. Theseagents are useful for treating conditions such as pruritus due tourticaria, atopic dermatitis, contact dermatitis, psoriasis, and manyothers. Examples of such reagents include, for example, terfenadine,astemizole, lorotadine, cetirizine, acrivastine, temelastine,cimetidine, ranitidine, famotidine, nizatidine, and the like. Tricyclicantidepressants can also be delivered using the delivery-enhancingtransporters of the invention.

[0209] Topical antipsoriasis drugs are also of interest. Agents such ascorticosteroids, calcipotriene, and anthralin can be conjugated to thedelivery-enhancing transporters of the invention and applied to skin.

[0210] The delivery-enhancing transporters of the invention are alsouseful for enhancing delivery of photochemotherapeutic agents into andacross one or more layers of skin and other epithelial tissues. Suchcompounds include, for example, the psoralens, and the like. Sunscreencomponents are also of interest; these include p-aminobenzoic acidesters, cinnamates and salicylates, as well as benzophenones,anthranilates, and avobenzone.

[0211] Pain relief agents and local anesthetics constitute another classof compounds for which conjugation to the delivery-enhancingtransporters of the invention can enhance treatment. Lidocaine,bupibacaine, novocaine, procaine, tetracaine, benzocaine, cocaine, andthe opiates, are among the compounds that one can conjugate to thedelivery-enhancing transporters of the invention. Application of painrelief agents to the joints or skin near the at the joints, e.g., inpatients suffering from rhematoid arthritis, is also contemplated.

[0212] Other biological agents of interest include, for example,minoxidil, keratolytic agents, destructive agents such as podophyllin,hydroquinone, capsaicin, masoprocol, colchicine, and gold.

[0213] Treatment of inflammed joints such as occurs in rhematoidarthritis can also be treated with compounds useful for such treatmentsconjugated to the transporters of the invention.

[0214] B. Gastrointestinal Administration

[0215] The delivery-enhancing transporters of the invention are alsouseful for delivery of conjugated drugs by gastrointestinaladministration. Gastrointestinal administration can be used for bothsystemically active drugs, and for drugs that act in thegastrointestinal epithelium.

[0216] Among the gastrointestinal conditions that are treatable usingappropriate reagents conjugated to the delivery-enhancing transportersare inflammatory bowel disease such as Crohn's disease (e.g.,cyclosporin and ascomycins such as FK506; aminosalicylates, e.g.,aspirin-like drugs, which include sulfasalazine and mesalamine;corticosteroids, e.g., prednisone and methylprednisolone; immunemodifiers, e.g., azathioprine, 6MP, methotrexate; and antibiotics, e.g.,metronidazole, ampicillin, ciprofloxacin, and others). Other treatablegastrointestinal conditions include ulcerative colitis, gastrointestinalulcers, peptic ulcer disease, imbalance of salt and water absorption(can lead to constipation, diarrhea, or malnutrition), abnormalproliferative diseases, and the like. Ulcer treatments include, forexample, drugs that reduce gastric acid secretion, such as H₂ histamineinhibitors (e.g., cymetidine and ranitidine) and inhibitors of theproton-potassium ATPase (e.g., lansoprazle and omeprazle), andantibiotics directed at Helicobacter pylori.

[0217] Compounds useful for the treatment of constipation can also beused in conjunction with the transporters of the invention. Usefulcompounds for treating constipation include, e.g., surfactant laxativessuch as docusate sodium, poloxamer 188, dehydrochloric acid andricinoleic acid. Exemplary stimulant laxatives include, e.g.,phenolphthalein, bisacodyl and anthraquinone laxatives such as danthron.

[0218] Antibiotics are among the biologically active agents that areuseful when conjugated to the delivery-enhancing transporters of theinvention, particularly those that act on invasive bacteria, such asShigella, Salmonella, and Yersinia. Such compounds include, for example,norfloxacin, ciprofloxacin, trimethoprim, sulfamethyloxazole, and thelike.

[0219] Anti-neoplastic agents, for example, for the treatment of coloncancer, can also be conjugated to the delivery-enhancing transporters ofthe invention and administered by the gastrointestinal route. Theseinclude, for example, cisplatin, methotrexate, taxol, fluorouracil,mercaptopurine, donorubicin, bleomycin, streptozocin, mitomycin and thelike.

[0220] For gastrointestinal and colonic delivery of orally administeredtransporters or active compounds, it can be beneficial to coat orencapsulate the compounds so that the compounds are not released untilthey are delivered to the gastrointestinal (GI) tract or colon. Methodsand composition useful for delivery to the GI tract or colon aredescribed in, e.g., U.S. Pat. Nos. 6,183,466 and 6,120,803.

[0221] C. Respiratory Tract Administration

[0222] The delivery-enhancing transporters of the invention can alsoused to enhance administration of drugs through the respiratory tract.The respiratory tract, which includes the nasal mucosa, hypopharynx, andlarge and small airway structures, provides a large mucosal surface fordrug absorption. The enhanced penetration of the conjugated agents intoand across one or more layers of the epithelial tissue that is providedby the delivery-enhancing transporters of the invention results inamplification of the advantages that respiratory tract delivery has overother delivery methods. For example, lower doses of an agent are oftenneeded to obtain a desired effect, a local therapeutic effect can occurmore rapidly, and systemic therapeutic blood levels of the agent areobtained quickly. Rapid onset of pharmacological activity can resultfrom respiratory tract administration. Moreover, respiratory tractadministration generally has relatively few side effects.

[0223] The transporters of the invention can be used to deliverbiological agents that are useful for treatment of pulmonary conditions.Examples of conditions treatable by nasal administration include, forexample, asthma. These compounds include antiinflammatory agents, suchas corticosteroids, cromolyn, and nedocromil, bronchodialators such asβ2-selective adronergic drugs and theophylline, and immunosuppressivedrugs (e.g., cyclosporin and FK506). Other conditions include, forexample, allergic rhinitis (which can be treated with glucocorticoids),and chronic obstructive pulmonary disease (emphysema). Other drugs thatact on the pulmonary tissues and can be delivered using the transportersof the invention include beta-agonists, mast cell stabilizers,antibiotics, antifungal and antiviral agents, surfactants, vasoactivedrugs, sedatives and hormones.

[0224] Respiratory tract administration is useful not only for treatmentof pulmonary conditions, but also for delivery of drugs to distanttarget organs via the circulatory system. A wide variety of such drugsand diagnostic agents can be administered through the respiratory tractafter conjugation to the delivery-enhancing transporters of theinvention.

[0225] D. Delivery of Agents across the Blood Brain Barrier

[0226] The delivery-enhancing transporters are also useful fordelivering biologically active and diagnostic agents across the bloodbrain barrier. The agents are useful for treating ischemia (e.g., usingan anti-apoptotic drug), as well as for delivering neurotransmitters andother agents for treating various conditions such as schizophrenia,Parkinson's disease, pain (e.g., morphine, the opiates). The5-hydroxytryptamine receptor antagonist is useful for treatingconditions such as migraine headaches and anxiety.

[0227] E. Diagnostic Imaging and Contrast Agents

[0228] The delivery-enhancing transporters of the invention are alsouseful for delivery of diagnostic imaging and contrast agents into andacross one or more layers of an epithelial and/or endothelial tissue.Examples of diagnostic agents include substances that are labeled withradioactivity, such as ⁹⁹mTc glucoheptonate, or substances used inmagnetic resonance imaging (MRI) procedures such as gadolinium dopedchelation agents (e.g. Gd-DTPA). Other examples of diagnostic agentsinclude marker genes that encode proteins that are readily detectablewhen expressed in a cell (including, but not limited to,(β-galactosidase, green fluorescent protein, luciferase, and the like. Awide variety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc.

[0229] F. Ocular Administration

[0230] The delivery-enhancing transporters of the invention can alsoused to enhance administration of drugs through the tissues of the eyeand other related tissues such as the eye lid. The ocular tissuesinclude the cornea, iris, lens, vitreus, vitreus humor, the optic nerveand the eyelid.

[0231] Examples of conditions treatable with the compositions of theinvention include the following. Conjunctivitis, sometimes called pinkeye, is an inflammation of the blood vessels in the conjunctiva, themembrane that covers the sclera and inside of the eyelids.Conjunctivitis may be caused by bacteria or viruses.

[0232] Antibacterial and antiviral compounds usful for treatingbacterial or viral infections of the eye are well known. See, e.g.,Hardman and Limbird, supra. Exemplary compounds include chloramphenicol,Ciproflaxacin, polymyxin B and tetracycline. Exemplary antiviralcompounds include idoxuridine, acyclovir and ganciclovir.

[0233] Styes are noncontagious, bacterial infections of one of thesebaceous glands of the eyelid. A stye looks like a small, red bumpeither on the eyelid or on the edge of the eyelid.

[0234] Dry eye, or “Sjögren's syndrome,” is an immune system disordercharacterized by inflammation and dryness of the mouth, eyes, and othermucous membranes, damages the lacrimal glands, and this damage affectstear production. Dry eye can be treated with immunosuppressive compoundssuch as cyclosporin as well as with ascomycins or steroids.

[0235] Glaucoma is a condition in which the normal fluid pressure insidethe eyes (intraocular pressure, or IOP) slowly rises as a result of thefluid aqueous humor, which normally flows in and out of the eye, notbeing able to drain properly. Instead, the fluid collects and causespressure damage to the optic nerve and loss of vision. Useful compoundsto treat glaucoma, blindness, and other eye disorders include, e.g.,timolol, levobunolol and phenylepherine. Growth factors such as nervegrowth factor (NGF) (see, e.g., Bennett, et al. Mol Ther 1(6):501-5(2000)) are also useful for treating glaucoma and other oculardisorders.

[0236] Therapeutic compounds for treatment of ocular diseases, such asthose discussed above, are well known to those of skill in the art.Typically, administration of the composition of the invention to theocular tissues is in the form of an eye drop. Alternatively, forexample, the compositions can be injected into the eye or applied as anointment.

[0237] Eye drops including the compounds of the invention can alsoinclude an isotonic agent added to sterilized purified water, and ifrequired, a preservative, a buffering agent, a stabilizer, a viscousvehicle and the like are added to the solution and dissolved therein.After dissolution, the pH is adjusted with a pH controller to be withina range suitable for use as an ophthalmic medicine, preferably withinthe range of 4.5 to 8.

[0238] Sodium chloride, glycerin, mannitol or the like may be used asthe isotonic agent; p-hydroxybenzoic acid ester, benzalkonium chlorideor the like as the preservative; sodium hydrogenphosphate, sodiumdihydrogenphosphate, boric acid or the like as the buffering agent;sodium edetate or the like as the stabilizer; polyvinyl alcohol,polyvinyl pyrrolidone, polyacrylic acid or the like as the viscousvehicle; and sodium hydroxide, hydrochloric acid or the like as the pHcontroller

[0239] Biologically Active and Diagnostic Molecules Useful with theDelivery-Enhancing Transporters

[0240] The delivery-enhancing transporters can be conjugated to a widevariety of biologically active agents and molecules that have diagnosticuse.

[0241] A. Small Organic Molecules

[0242] Small organic molecule therapeutic agents can be advantageouslyattached to linear polymeric compositions as described herein, tofacilitate or enhance transport across one or more layers of anepithelial or endothelial tissue. For example, delivery of highlycharged agents, such as levodopa (L-3,4-dihydroxy-phenylalanine; L-DOPA)may benefit by linkage to delivery-enhancing transporters as describedherein. Peptoid and peptidomimetic agents are also contemplated (e.g.,Langston (1997) DDT2:255; Giannis et al. (1997) Advances Drug Res.29:1). Also, the invention is advantageous for delivering small organicmolecules that have poor solubilities in aqueous liquids, such as serumand aqueous saline. Thus, compounds whose therapeutic efficacies arelimited by their low solubilities can be administered in greater dosagesaccording to the present invention, and can be more efficacious on amolar basis in conjugate form, relative to the non-conjugate form, dueto higher uptake levels by cells.

[0243] Since a significant portion of the topological surface of a smallmolecule is often involved, and therefore required, for biologicalactivity, the small molecule portion of the conjugate in particularcases may need to be severed from the attached delivery-enhancingtransporter and linker moiety (if any) for the small molecule agent toexert biological activity after crossing the target epithelial tissue.For such situations, the conjugate preferably includes a cleavablelinker for releasing free drug after passing through an epithelialtissue.

[0244]FIG. 5D and FIG. 5E are illustrative of another aspect of theinvention, comprising taxane- and taxoid anticancer conjugates whichhave enhanced trans-epithelial tissue transport rates relative tocorresponding non-conjugated forms. The conjugates are particularlyuseful for inhibiting growth of cancer cells. Taxanes and taxoids arebelieved to manifest their anticancer effects by promotingpolymerization of microtubules (and inhibiting depolymerization) to anextent that is deleterious to cell function, inhibiting cell replicationand ultimately leading to cell death.

[0245] The term “taxane” refers to paclitaxel (FIG. 5F, R′=acetyl,R″=benzyl) also known under the trademark “TAXOL”) and naturallyoccurring, synthetic, or bioengineered analogs having a backbone corethat contains the A, B, C and D rings of paclitaxel, as illustrated inFIG. 5G. FIG. 5F also indicates the structure of “TAXOTERE™” (R′=H,R″=BOC), which is a somewhat more soluble synthetic analog of paclitaxelsold by Rhone-Poulenc. “Taxoid” refers to naturally occurring, syntheticor bioengineered analogs of paclitaxel that contain the basic A, B and Crings of paclitaxel, as shown in FIG. 5H. Substantial synthetic andbiological information is available on syntheses and activities of avariety of taxane and taxoid compounds, as reviewed in Suffness (1995)Taxol: Science and Applications, CRC Press, New York, N.Y., pp. 237-239,particularly in Chapters 12 to 14, as well as in the subsequentpaclitaxel literature. Furthermore, a host of cell lines are availablefor predicting anticancer activities of these compounds against certaincancer types, as described, for example, in Suffness at Chapters 8 and13.

[0246] The delivery-enhancing transporter is conjugated to the taxane ortaxoid moiety via any suitable site of attachment in the taxane ortaxoid. Conveniently, the transport polymer is linked via a C2′-oxygenatom, C7-oxygen atom, using linking strategies as above. Conjugation ofa transport polymer via a C7-oxygen leads to taxane conjugates that haveanticancer and antitumor activity despite conjugation at that position.Accordingly, the linker can be cleavable or non-cleavable. Conjugationvia the C2′-oxygen significantly reduces anticancer activity, so that acleavable linker is preferred for conjugation to this site. Other sitesof attachment can also be used, such as C10.

[0247] It will be appreciated that the taxane and taxoid conjugates ofthe invention have improved water solubility relative to taxol (≈0.25μg/mL) and taxotere (6-7 μg/mL). Therefore, large amounts ofsolubilizing agents such as “CREMOPHOR EL” (polyoxyethylated castoroil), polysorbate 80 (polyoxyethylene sorbitan monooleate, also known as“TWEEN 80”), and ethanol are not required, so that side-effectstypically associated with these solubilizing agents, such asanaphylaxis, dyspnea, hypotension, and flushing, can be reduced.

[0248] B. Metals

[0249] Metals can be transported into and across one or more layers ofepithelial and endothelial tissues using chelating agents such astexaphyrin or diethylene triamine pentacetic acid (DTPA), conjugated toa delivery-enhancing transporter of the invention, as illustrated byExample . These conjugates are useful for delivering metal ions forimaging or therapy. Exemplary metal ions include Eu, Lu, Pr, Gd, Tc99m,Ga67, In111, Y90, Cu67, and Co57. Preliminary membrane-transport studieswith conjugate candidates can be performed using cell-based assays suchas described in the Example section below. For example, using europiumions, cellular uptake can be monitored by time-resolved fluorescencemeasurements. For metal ions that are cytotoxic, uptake can be monitoredby cytotoxicity.

[0250] C. Macromolecules

[0251] The enhanced transport methods of the invention are particularlysuited for enhancing transport into and across one or more layers of anepithelial or endothelial tissue for a number of macromolecules,including, but not limited to proteins, nucleic acids, polysaccharides,and analogs thereof. Exemplary nucleic acids include oligonucleotidesand polynucleotides formed of DNA and RNA, and analogs thereof, whichhave selected sequences designed for hybridization to complementarytargets (e.g., antisense sequences for single- or double-strandedtargets), or for expressing nucleic acid transcripts or proteins encodedby the sequences. Analogs include charged and preferably unchargedbackbone analogs, such as phosphonates (preferably methyl phosphonates),phosphoramidates (N3′ or N5′), thiophosphates, unchargedmorpholino-based polymers, and protein nucleic acids (PNAs). Suchmolecules can be used in a variety of therapeutic regimens, includingenzyme replacement therapy, gene therapy, and anti-sense therapy, forexample.

[0252] By way of example, protein nucleic acids (PNA) are analogs of DNAin which the backbone is structurally homomorphous with a deoxyribosebackbone. The backbone consists of N-(2-aminoethyl)glycine units towhich the nucleobases are attached. PNAs containing all four naturalnucleobases hybridize to complementary oligonucleotides obeyingWatson-Crick base-pairing rules, and is a true DNA mimic in terms ofbase pair recognition (Egholm et al. (1993) Nature 365:566-568. Thebackbone of a PNA is formed by peptide bonds rather than phosphateesters, making it well-suited for anti-sense applications. Since thebackbone is uncharged, PNA/DNA or PNA/RNA duplexes that form exhibitgreater than normal thermal stability. PNAs have the additionaladvantage that they are not recognized by nucleases or proteases. Inaddition, PNAs can be synthesized on an automated peptides synthesizerusing standard t-Boc chemistry. The PNA is then readily linked to atransport polymer of the invention.

[0253] Examples of anti-sense oligonucleotides whose transport into andacross epithelial and endothelial tissues can be enhanced using themethods of the invention are described, for example, in U.S. Pat. No.5,594,122. Such oligonucleotides are targeted to treat humanimmunodeficiency virus (HIV). Conjugation of a transport polymer to ananti-sense oligonucleotide can be effected, for example, by forming anamide linkage between the peptide and the 5′-terminus of theoligonucleotide through a succinate linker, according towell-established methods. The use of PNA conjugates is furtherillustrated in Example 11 of PCT Application PCT/US98/10571. FIG. 7 ofthat application shows results obtained with a conjugate of theinvention containing a PNA sequence for inhibiting secretion ofgamma-interferon (γ-IFN) by T cells, as detailed in Example 11. As canbe seen, the anti-sense PNA conjugate was effective to block γ-IFNsecretion when the conjugate was present at levels above about 10 μM. Incontrast, no inhibition was seen with the sense-PNA conjugate or thenon-conjugated antisense PNA alone.

[0254] Another class of macromolecules that can be transported acrossone or more layers of an epithelial or endothelial tissue is exemplifiedby proteins, and in particular, enzymes. Therapeutic proteins include,but are not limited to replacement enzymes. Therapeutic enzymes include,but are not limited to, alglucerase, for use in treating lysozomalglucocerebrosidase deficiency (Gaucher's disease), alpha-L-iduronidase,for use in treating mucopolysaccharidosis I,alpha-N-acetylglucosamidase, for use in treating sanfilippo B syndrome,lipase, for use in treating pancreatic insufficiency, adenosinedeaminase, for use in treating severe combined immunodeficiencysyndrome, and triose phosphate isomerase, for use in treatingneuromuscular dysfunction associated with triose phosphate isomerasedeficiency.

[0255] In addition, and according to an important aspect of theinvention, protein antigens may be delivered to the cytosoliccompartment of antigen-presenting cells (APCs), where they are degradedinto peptides. The peptides are then transported into the endoplasmicreticulum, where they associate with nascent HLA class I molecules andare displayed on the cell surface. Such “activated” APCs can serve asinducers of class I restricted antigen-specific cytotoxic T-lymphocytes(CTLs), which then proceed to recognize and destroy cells displaying theparticular antigen. APCs that are able to carry out this processinclude, but are not limited to, certain macrophages, B cells anddendritic cells. In one embodiment, the protein antigen is a tumorantigen for eliciting or promoting an immune response against tumorcells. The transport of isolated or soluble proteins into the cytosol ofAPC with subsequent activation of CTL is exceptional, since, with fewexceptions, injection of isolated or soluble proteins does not resulteither in activation of APC or induction of CTLs. Thus, antigens thatare conjugated to the transport enhancing compositions of the presentinvention may serve to stimulate a cellular immune response in vitro orin vivo.

[0256] In another embodiment, the invention is useful for deliveringimmunospecific antibodies or antibody fragments to the cytosol tointerfere with deleterious biological processes such as microbialinfection. Recent experiments have shown that intracellular antibodiescan be effective antiviral agents in plant and mammalian cells (e.g.,Tavladoraki et al. (1993) Nature 366:469; and Shaheen et al. (1996) J.Virol. 70:3392. These methods have typically used single-chain variableregion fragments (scFv), in which the antibody heavy and light chainsare synthesized as a single polypeptide. The variable heavy and lightchains are usually separated by a flexible linker peptide (e.g., of 15amino acids) to yield a 28 kDa molecule that retains the high affinityligand binding site. The principal obstacle to wide application of thistechnology has been efficiency of uptake into infected cells. But byattaching transport polymers to scFv fragments, the degree of cellularuptake can be increased, allowing the immunospecific fragments to bindand disable important microbial components, such as HIV Rev, HIV reversetranscriptase, and integrase proteins.

[0257] D. Peptides

[0258] Peptides to be delivered by the enhanced transport methodsdescribed herein include, but should not be limited to, effectorpolypeptides, receptor fragments, and the like. Examples includepeptides having phosphorylation sites used by proteins mediatingintra-cellular signals. Examples of such proteins include, but are notlimited to, protein kinase C, RAF-1, p21Ras, NF-κB, C-JUN, andcytoplasmic tails of membrane receptors such as IL-4 receptor, CD28,CTLA-4, V7, and MHC Class I and Class II antigens.

[0259] When the delivery-enhancing transporter is also a peptide,synthesis can be achieved either using an automated peptide synthesizeror by recombinant methods in which a polynucleotide encoding a fusionpeptide is produced, as mentioned above.

EXAMPLES

[0260] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1 Penetration of Biotinylated Polymers of D-Arginine into theSkin of Nude Mice

[0261] This Example demonstrates that poly-arginine heptamers candeliver conjugated biotin into and across layers of the skin, bothfollicularly and interfollicularly, and into the dermis.

[0262] Methods

[0263] Biotinylated peptides were synthesized using solid phasetechniques and commercially available Fmoc amino acids, resins, andreagents (PE Biosystems, Foster City Calif., and Bachem Torrence,Calif.) on a Applied Biosystems 433 peptide synthesizer. Fastmoc cycleswere used with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexfluorophosphate (HATU) substituted for HBTU/HOBt as the couplingreagent. Prior to the addition of biotin to the amino terminus of thepeptide, amino caproic acid (aca) was conjugated and acted as a spacer.The peptides were cleaved from the resin using 96% trifluoroacetic acid,2% triisopropyl silane, and 2% phenol for between 1 and 12 hours. Thelonger reaction times were necessary to completely remove the Pbfprotecting groups from the polymers of arginine. The peptidessubsequently were filtered from the resin, precipitated using diethylether, purified using HPLC reverse phase columns (Alltech Altima,Chicago, Ill.) and characterized using either electrospray or matrixassisted laser desorption mass spectrometry (Perceptive Biosystems,Boston, Mass.).

[0264] Varying concentrations (1 mM-10 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in phosphate buffered saline(PBS), were applied to the back of anesthetized nude mice. Samples (100μl) were applied as a liquid without excipient, prevented fromdispersing by a Vaseline™ barrier, and allowed to penetrate for fifteenminutes. At the end of this period the animal was sacrificed, therelevant sections of skin were excised, embedded in mounting medium(OCT), and frozen. Frozen sections (5 microns) were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.). The slides werefixed in acetone at 4° C. for ten minutes, air dried, soaked in PBS forfive minutes, blocked with normal goat serum for five minutes, andwashed with PBS for five minutes. The section was stained by incubationwith fluorescently labeled streptavidin at 30 μg/ml for thirty minutes,washed with PBS, counterstained with propidium iodide (1 μg/ml) for twominutes, and the section was mounted with Vectashield™ mounting media.Slides were analyzed by fluorescent microscopy. Parallel studies weredone using streptavidin-horse radish peroxidase rather thanfluorescein-streptavidin. The biotinylated peptide was visualized bytreatment of the sections with the horseradish peroxidase substratediaminobenzadine, and visualization with light microscopy.

[0265] Results

[0266] Biotinylated arginine heptamer crossed into and across theepidermis and into the dermis. The cytosol and nuclei of all cells inthe field were fluorescent, indicating penetration into virtually everycell of the nude mouse skin in the section. The staining pattern wasconsistent with unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. In contrast, no staining was apparent in micetreated with biotin alone, or phosphate buffered saline alone.

Example 2 Penetration of Biotinylated Polymers of D-Arginine into theSkin of Normal Balb/C Mice

[0267] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, were applied to a skinof the groin of an anesthetized Balb/C mice. Sample (100 μl ) wasapplied as a liquid within excipient and prevented from dispersing by aVaseline™ barrier and allowed to penetrate for thirty minutes. At theend of this period animal was sacrificed, the relevant section of skinwas excised, embedded in mounting medium (OCT) and frozen. Frozensections were cut using a cryostat, collected on slides, and stainedwith fluorescently labeled streptavidin (Vector Laboratories,Burlingame, Calif.) as described in Example 1. Slides were analyzed byfluorescent microscopy.

[0268] Results

[0269] As with the skin from nude mice, biotinylated arginine heptamercrossed into and across the epidermis and into the dermis. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into virtually every cell of the nude mouse skin in thesection. The staining pattern was consistent with unanticipatedtransport that was both follicular and interfollicular. In addition,positive cells were apparent in papillary and reticular dermis. Incontrast, no staining was apparent in mice treated with biotin alone, orphosphate buffered saline alone.

Example 3 Penetration of Biotinylated Polymers of D-Arginine into NormalHuman Skin Grafted onto Nude Mice

[0270] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, were applied to humanforeskin grafts on the back of SCID mice (see, e.g., Deng et al. (1997)Nature Biotechnol. 15: 1388-1391; Khavari et al. (1997) Adv. Clin. Res.15:27-35; Choate and Khavari (1997) Human Gene Therapy 8:895-901).Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forfifteen minutes. At the end of this period animal was sacrificed, therelevant section of skin was excised, embedded in mounting medium (OCT)and frozen. Frozen sections were cut using a cryostat, collected onslides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

[0271] Results

[0272] As with the skin from nude and normal mice, biotinylated arginineheptamer crossed into and across the epidermis and into the dermis ofthe human skin. The cytosol and nuclei of all cells in the field werefluorescent, indicating penetration into and through the epidermis anddermis. Intense staining was seen at both 20× and 40× magnification. Thestaining pattern was consistent with unanticipated transport that wasboth follicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. In contrast, no staining wasapparent in mice treated with biotin alone, or phosphate buffered salinealone, and very little staining was observed with the biotinylatedarginine pentamer conjugate, either at low or high magnification.

Example 4 Increased Penetration of Biotinylated Polymers of D-Arginineinto Skin of Nude Mouse Using Plastic Wrap or a Lotion Excipient

[0273] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, and mixed with an equalvolume of Lubridern™. The lotion mixture was then applied to the back ofnude mice and allowed to penetrate for thirty, sixty, and 120 minutes.Alternatively, sample (100 μl) was applied as a liquid without excipientand prevented from evaporating by wrapping plastic wrap over the samplesealed with Vaseline™. The samples were allowed to penetrate for thirty,sixty, and 120 minutes. At the end of this period animal was sacrificed,the relevant section of skin was excised, embedded in mounting medium(OCT) and frozen. Frozen sections were cut using a cryostat, collectedon slides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

[0274] Results

[0275] Both lotion and plastic wrap resulted in increased uptakecompared with staining without excipient. Lotion was more effective thanplastic wrap in enhancing uptake of the conjugate. Biotinylated argininepentamers crossed into and across several skin layers, reaching both thecytosol and nuclei of epidermal cell layers, both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis.

Example 5 Penetration of Cyclosporin Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice

[0276] Methods

[0277] A. Linking Cyclosporin to Delivery-Enhancing Transporters

[0278] 1. Preparation of the α-Chloroacetyl Cyclosporin A Derivative.

[0279] The α-chloroacetyl cyclosporin A derivative was prepared as shownin FIG. 1. Cyclosporin A (152.7 mg, 127 μmol) and chloroacetic acidanhydride (221.7 mg; 1300 μmol) were placed into a dry flask underN₂-atmosphere. Pyridine (1.0 mL) was added and the solution was heatedto 50° C. (oil bath). After 16 hours the reaction was cooled to roomtemperature and quenched with water (4.0 mL). The resulting suspensionwas extracted with diethylether (Σ 15 mL). The combined organic layerswere dried over MgSO₄. Filtration and evaporation of solvents in vacuodelivered a yellow oil, which was purified by flash chromatography onsilica gel (eluent: EtOAc/hexanes: 40%-80%) yielding 136 mg (106.4 μmol,83%) of the desired product.

[0280] 2. Coupling to Transporter Molecules

[0281] A general procedure for the coupling of cysteine containingpeptides to the α-chloro acetyl Cyclosporin A derivative is shown inFIG. 2.

[0282] a. Labeled Peptides

[0283] The cyclosporin A derivative and the labeled peptide (1equivalent) were dissolved in DMF (10 mmol of Cyclosporin Aderivative/mL DMF) under an N₂-atmosphere. Diisopropylethylamine (10equivalents) was added and stirring at room temperature was continueduntil all starting material was consumed (usually after 16 hours) (FIG.3). The solvents were removed in vacuo and the crude reaction productwas dissolved in water and purified by reversed phase high pressureliquid chromatography (RP-HPLC) (eluent:water/MeCN*TFA). The productswere obtained in the following yields:

[0284] B-aca-r5-Ala-Ala-Cys-O-acyl-Cyclosporin A: 47%

[0285] B-aca-r7-Cys-O-acyl-Cyclosporin A: 43%

[0286] B-aca-r9-Cys-O-acyl-Cyclosporin A: 34%

[0287] B-aca-Cys-O-acyl-Cyclosporin A: 55%

[0288] b. Unlabeled Peptides

[0289] The peptide (34.7 mg, 15.3 μmol) and the Cyclosporin A derivative(19.6 mg, 15.3 μmol) were dissolved in DMF (1.0 mL) under anN₂-atmosphere (FIG. 4). Diisopropylethylamine (19.7 mg, 153 μmol) wasadded and stirring at room temperature was continued. After 12 hours thesolvent was removed in vacuo. The crude material was dissolved in waterand purified by RP-HPLC (eluent:water/MeCN*TFA) yielding the pureproduct (24.1 mg, 6.8 mmol, 44%).

[0290] B. Analysis of Transport Across Skin

[0291] Varying concentrations (1 mM-100 μM) of cyclosporin conjugated toeither biotinylated pentamer, heptamer, or nonamers of D-arginine (bior5, r7, or r9), dissolved in PBS, were applied to the back of nude mice.Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a VaselineTm barrier and allowed to penetrate forthirty, sixty, and 120 minutes. At the end of this period animal wassacrificed, the relevant section of skin was excised, embedded inmounting medium (OCT) and frozen. Frozen sections were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.) as described inExample 1. Slides were analyzed by fluorescent microscopy

[0292] Results

[0293] The conjugates of cyclosporin with biotinylated heptamers andnonamers of D-arginine effectively entered into and across the epidermisand into the dermis of the skin of nude mice. In contrast, very littleuptake was seen using a conjugate between a pentamer of arginine andcyclosporin, and no staining was seen with a PBS control. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into and through the epidermis and dermis. The stainingpattern was consistent with unanticipated transport that was bothfollicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. These results demonstrateremarkable uptake only when sufficient guanidinyl groups are included inthe delivery-enhancing transporter.

Example 6 Demonstration that a D-Arginine Heptamer Can Penetrate HumanSkin

[0294] Human and murine skin differ significantly in a number of ways,with human epidermis being considerably thicker. To determine if theD-arginine heptamers/cyclosporin A (r7 CsA) conjugate could alsopenetrate human skin, biotin r7 CsA was applied to full thickness humanskin grafted onto the back of a SCID mouse. As in murine skin,conjugated cyclosporin A penetrated human epidermis and dermis.Fluorescence was observed in both the cytosol and the nuclei of cells intissue exposed to biotinylated peptides alone, but in sections stainedwith biotin r7 CsA the majority of fluorescence was cytosolic,consistent with r7 CsA binding to cyclosporin A's known cytoplasmictargets.

Example 7 Demonstration that Cyclosporin A-Transporter Conjugates EnterT Cells in the Dermis

[0295] Methods

[0296] Inhibition of IL-2 Secretion by Releasable R7-CsA Conjugate.

[0297] Jurkat cells (5×10⁴) were incubated with varying concentrationsof a nonreleasable or releasable R7-CsA conjugate or CsA overnight at37° C. to allow for the release of the active form of CsA prior tostimulation with PMA and ionomycin. T cells subsequently were stimulatedto produce IL-2 by addition of 10 ng/ml PMA (Sigma, St. Louis, Mo.) and1 μM ionomycin (CalBiochem, San Diego, Calif.). Cultures were incubatedovernight at 37° C. and supernatants were collected and IL-2 wasmeasured using a fluorescent ELISA. Briefly, plates were coated with 4μg/ml anti-human IL-2 antibody (BD Pharmingen, San Diego, Calif.),blocked with PBS containing 10% FBS for 1 hour at room temperature,washed, and supernatants added and incubated for 1 hour. Media wasremoved and biotinylated anti-human IL-2 (1.6 μg/ml), was added for onehour. The plates were washed, and then europium labeled streptavidin(0.04 ng/ml) was added for one hour. After another wash, enhancementsolution was added and the resulting fluorescence was measured using aWallac plate reader (Wallac, Turku, Finland).

[0298] Results

[0299] To determine whether biotinylated D-arginine heptamer-cyclosporin(r7 CsA) conjugate would reach infiltrating T cells within inflamed skinin vivo, biotin r7 CsA was applied to the site of inflammation on theback of a mouse with experimentally induced contact dermatitis. Inflamedskin was stained with rhodamine labeled goat anti-mouse CD3 to localizeT-cells and with fluorescein labeled streptavidin to localize the biotinr7 CsA. Biotin r7 CsA was found in all CD3[+] T cells in the tissue inaddition to a variety of other cells that probably represent otherinflammatory cells as well as resident fibroblasts. These data indicatethat biotin r7 CsA penetrates inflamed skin to reach key target Tlymphocytes.

Example 8 Synthesis, in vitro and in vivo Activity of a ReleasableConjugate of a Short Oligomer of Arginine and CsA

[0300] Modification of the 20 alcohol of Cyclosporin A results insignificant loss of its biological activity. See, e.g., R. E.Handschumacher, et al, Science 226, 544-7 (1984). Consequently, toensure release of free Cyclosporin A from its conjugate after transportinto cells, Cyclosporin A was conjugated to an oligo-argininetransporter through a pH sensitive linker as shown in FIG. 10. Theresultant conjugate is stable at acidic pH but at pH>7 it undergoes anintramolecular cyclization involving addition of the free amine to thecarbonyl adjacent to Cyclosporin A (FIG. 6), which results in therelease of unmodified Cyclosporin A.

[0301] Another modification in the design of the releasable conjugatewas the use of L-arginine (R), and not D-arginine (r) in thetransporter. While the oligo-D-arginine transporters were used for thehistological experiments to ensure maximal stability of the conjugateand therefore accuracy in determining its location through fluorescence,oligomers of L-arginine were incorporated into the design of thereleasable conjugate to minimize its biological half-life. Consistentwith its design, the resultant releasable conjugate was shown to bestable at acidic pH, but labile at physiological pH in the absence ofserum. This releasable Cyclosporin A conjugate's half-life in pH 7.4 PBSwas 90 minutes.

[0302] Results

[0303] The releasable guanidino-heptamer conjugate of Cyclosporin A wasshown to be biologically active by inhibiting IL-2 secretion by thehuman T cell line, Jurkat, stimulated with PMA and ionomycin in vitro.See R. Wiskocil, et al., J Immunol 134, 1599-603 (1985). The conjugatewas added 12 hours prior to the addition of PMA/ionomycin and dosedependent inhibition was observed by the releasable R7 CsA conjugate.This inhibition was not observed with a nonreleasable analog (FIG. 6)that differed from the releasable conjugate by retention of the t-Bocprotecting group, which prevented cyclization and resultant release ofthe active drug. The EC₅₀ of the releasable R7 cyclosporin conjugate wasapproximately two fold higher than CsA dissolved in alcohol and added atthe same time as the releasable conjugate.

[0304] The releasable R7 CsA conjugate was assayed in vivo forfunctional activity using a murine model of contact dermatitis.Treatment with the 1% releasable R7 CSA conjugate resulted in 73.9%±4.0reduction in ear inflammation (FIG. 7). No reduction in inflammation wasseen in the untreated ear, indicating that the effect seen in thetreated ear was local and not systemic. Less inhibition was observed inthe ears of mice treated with 0.1 and 0.01% R7-CsA (64.8%±4.0 and 40.9%±3.3 respectively), demonstrating that the effect was titratable.Treatment with the fluorinated corticosteroid positive control resultedin reduction in ear swelling (34.1%±6.3), but significantly less thanthat observed for 0.1% releasable R7 CsA (FIG. 7). No reduction ofinflammation was observed in any of the mice treated with unmodifiedCyclosporin A, vehicle alone, R7, or nonreleasable R7 CsA.

Example 9 The Penetration of Copper and Gadolinium-DTPA-r7 Complexesinto the Skin of Nude Mice

[0305] Methods

[0306] 1. Preparation of Metal Complexes

[0307] Step 1—Preparation of Copper-diethylenetriaminepentaacetic AcidComplex (Cu-DTPA)

[0308] Copper carbonate (10 mmol) and diethylenetriaminpentacetic acid(10 nmol) were dissolved in water (150 mL) (FIG. 8). After 18 h, thesolution was centrifuged to removed any solids. The blue solution wasdecanted and lyophilized to provide a blue powder (yields >90%).

[0309] Step 2—Preparation of DTPA Transporter

[0310] The Cu-DTPA was linked to a transporter through an aminocaproicacid spacer using a PE Applied Biosystems Peptide Synthesizer (ABI 433A)(FIG. 9). The material was cleaved from the resin by treatment withtrifluoroacetic acid (TFA) (40 mL), triisopropyl silane (100 μL) andphenol (100 μL) for 18 h. The resin was filtered off and the peptide wasprecipitated by addition of diethyl ether (80 mL). The solution wascentrifuged and the solvent decanted off. The crude solid was purifiedby reverse-phase HPLC using a water/acetonitrile gradient. Treatmentwith TFA resulted in loss of Cu²⁺ ion which needed to be reinserted.

[0311] DTPA-aca-R7-CO2H (10 mg, 0.0063 mmol) and copper sulfate (1.6 mg,0.0063 mmol) were dissolved in water (1 mL). Let gently stir for 18 hand lyophilized to provide product as a white powder (10 mg).

[0312] 2. Analysis of Transport Across Skin

[0313] Metal diethylenetriaminepentaacetic acid (DTPA) complexes wereformed by mixing equimolar amounts of metal salts with DTPA in water for18 hours. At the end of this time, the solutions were centrifuged,frozen and lyophilized. The dried powder was characterized by massspectrometry and used in solid phase peptide synthesis. The metal-DTPAcomplexes were attached to polymers of D- or L-arginine that were stillattached to solid-phase resin used in peptide synthesis. The metal-DTPAcomplexes were attached using an aminocaproic acid spacer. The solidphase peptide synthesis techniques were described in Example 1, with theexception that cleavage of the peptide-DTPA-metal complex intrifluoroacetic acid released the metal. The metal is replaced afterHPLC purification and lyophilization of the peptide-DTPA complex.Replacement of the metal involved incubation of equimolar amounts of themetal salt with the peptide-aminocaproic acid-DTPA complex andsubsequent lyophilization.

[0314] Varying concentrations (1 μM to 1 mM) of the Cu-DTPA-aca-r7complex were applied to the abdominal region of nude mice for 15, 30 and45 minutes. As controls, an equimolar amount of the Cu-DTPA complex wasspotted onto the abdominal region. At the end of the incubation period,the samples were simply wiped off and intense blue color was apparent onthe skin where the Cu-DTPA-aca-r7 complex was spotted and not where theCu-DTPA alone was spotted. In the case of the application of 1 mM,visible blue dye was seen for three days, decreasing with time, butbeing apparent for the full period.

[0315] Varying concentrations (1 μM to 1 mM) of the Gd-DTPA-aca-r7complex are injected into the tail vein of BALB/c mice in 100 μl.Distribution of the Gd is observed in real time using magnetic resonanceimaging. Distribution of the dye is apparent throughout the bloodstream,entering liver, spleen, kidney, and heart. When injected into thecarotid artery of rabbits, the dye is seen to cross the blood brainbarrier.

Example 10 Penetration of Hydrocortisone Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice

[0316] Methods

[0317] A. Linking of Hydrocortisone to Delivery-Enhancing Transporters

[0318] Step 1—Acylation of Hydrocortisone with Chloroacetic Anhydride.

[0319] A solution of hydrocortisone (200 mg, 0.55 mmol) and chloroaceticanhydride (113 mg, 0.66 mmol) in pyridine (5 mL) was stirred at roomtemperature for 2 h (FIG. 10). The solvent was evaporated off and thecrude product was chromatographed on silica using 50% hexanes/ethylacetate as the eluent. Product isolated a whites solid (139 mg, 58%).

[0320] Step 2—Linking to Transporter.

[0321] A solution of the chloroacetic ester of hydrocortisone (0.0137mmol), a transporter containing a cysteine residue (0.0137) anddiisopropylethylamine (DIEA) (0.0274 mmol) in dimethylformamide (DMF) (1mL) was stirred at room temperature for 18 h (FIG. 11). The material waspurified via reverse-phase HPLC using a water/acetonitrile gradient andlyophilized to provide a white powder.

[0322] r5 conjugate—12 mg obtained (29% isolated yield)

[0323] r7 conjugate—22 mg obtained (55% isolated yield)

[0324] R7 conjugate—13 mg obtained (33% isolated yield)

[0325] B. Analysis of Transport Across Skin

[0326] Varying concentrations (1 mM-100 μM) of hydrocortisone conjugatedto either biotinylated pentamer, heptamer, or nonamers of D-arginine(bio r5, r7, or r9), dissolved in PBS, were applied to the back of nudemice. Samples (100 μl ) were applied as a liquid within excipient andprevented from dispersing by a Vaseline™ barrier and allowed topenetrate for thirty, sixty, and 120 minutes. At the end of this periodanimal was sacrificed, the relevant section of skin was excised,embedded in mounting medium (OCT) and frozen. Frozen sections were cutusing a cryostat, collected on slides, and stained with fluorescentlylabeled streptavidin (Vector Laboratories, Burlingame, Calif.) asdescribed in Example 1. Slides were analyzed by fluorescent microscopy.Results The conjugates of hydrocortisone with biotinylated heptamers ofD-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine andhydrocortisone, and no staining was seen with a PBS control. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into and through the epidermis and dermis. The stainingpattern was consistent with unanticipated transport that was bothfollicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. These results demonstrateremarkable uptake only when sufficient guanidinyl groups are included inthe delivery-enhancing transporter.

Example 11 Penetration of Taxol Conjugated to a Biotinylated Pentamer,Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice

[0327] Methods

[0328] 1. Conjugation of C-2′ Activated Taxol Derivatives toBiotin-Labeled Peptides

[0329] Synthesis of C-2′ Derivatives

[0330] Taxol (48.7 mg, 57.1 μmol) was dissolved in CH₂Cl₂ (3.0 mL) underan N₂-atmosphere. The solution was cooled to 0° C. A stock solution ofthe chloroformate of benzyl-(p-hydroxy benzoate) (200 mmol, in 2.0 mLCH₂Cl₂-freshly prepared from benzyl-(p-hydroxy benzoate) and diphosgene)was added at 0° C. and stirring at that temperature was continued for 5hours, after which the solution was warmed to room temperature (FIG.12). Stirring was continued for additional 10 hours. The solvents wereremoved in vacuo and the crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 30%-70%) yieldingthe desired taxol C-2′ carbonate (36.3 mg, 32.8 μmol, 57.4%).

[0331] Coupling to Biotin-Labeled Peptides.

[0332] A procedure for coupling to biotin-labeled peptides is shown inFIG. 13. The taxol derivative and the biotin labeled peptide (1.2equivalents) were dissolved in DMF (˜10 μmol/mL DMF) under anN₂-atmosphere. Stock solutions of diisopropylethylamine (1.2 equivalentsin DMF) and DMAP (0.3 equivalents in DMF) were added and stirring atroom temperature was continued until all starting material was consumed.After 16 hours the solvent was removed in vacuo. The crude reactionmixture was dissolved in water and purified by RP-HPLC(eluent:water/MeCN*TFA) yielding the conjugates in the indicated yields:

[0333] B-aca-r5-K-taxol: 3.6 mg, 1.32 mmol, 20%.

[0334] B-aca-r7-K-taxol: 9.8 mg, 3.01 mmol, 44%.

[0335] B-aca-r9-K-taxol: 19.4 mg, 5.1 mmol, 67%.

[0336] Unlabeled C-2′ Carbamates:

[0337] The taxol derivative (12.4 mg, 11.2 μmol) and the unlabeledpeptide (27.1 mg, 13.4 μmol) were dissolved in DMF (1.5 mL) under anN₂-atmosphere (FIG. 14). Diisopropylethylamine (1.7 mg, 13.4 μmol) wasadded as a stock solution in DMF, followed by DMAP (0.68 mg, 5.6 μmol)as a stock solution in DMF. Stirring at room temperature was continueduntil all starting material was consumed. After 16 hours the solvent wasremoved in vacuo. The crude material was dissolved in water and purifiedby RP-HPLC (eluent:water/MeCN*TFA) yielding the desired product (16.5mg, 5.9 μmol, 53%).

[0338] Other C-2′ Conjugates

[0339] The taxol derivative (8.7 mg, 7.85 μmol) was dissolved in EtOAc(2.0 mL). Pd/C (10%, 4.0 mg) was added and the reaction flask was purgedwith H₂ five times (FIG. 15A). Stirring under an atmosphere of hydrogenwas continued for 7 hours. The Pd/C was filtered and the solvent wasremoved in vacuo. The crude material (6.7 mg, 6.58 μmol, 84%) obtainedin this way was pure and was used in the next step without furtherpurification.

[0340] The free acid taxol derivative (18.0 mg, 17.7 μmol) was dissolvedin CH₂C1 ₂ (2.0 mL). Dicyclohexylcarbodiimide (4.3 mg, 21.3 μmol) wasadded as a stock solution in CH₂Cl₂ (0.1 mL). N-Hydroxysuccinimide (2.0mg, 17.7 μmol) was added as a stock solution in DMF (0.1 mL) (FIG. 15B).Stirring at room temperature was continued for 14 hours. The solvent wasremoved in vacuo and the resultant crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 40%-80%) yieldingthe desired product (13.6 mg, 12.2 μmol, 69%).

[0341] The activated taxol derivative (14.0 mg, 12.6 μmol) and thepeptide (30.6 mg, 15.1 μmol) were dissolved in DMF (3.0 mL) under anN₂-atmosphere (FIG. 15C). Diisopropylethylamine (1.94 mg, 15.1 μmol) wasadded as a stock solution in DMF (0.1 mL), followed by DMAP (0.76 mg,6.3 μmol) as a stock solution in DMF 0.1 mL). Stirring at roomtemperature was continued until all the starting material was consumed.After 20 hours the solvent was removed in vacuo. The crude material wasdissolved in water and purified by RP-HPLC (eluent:water/MeCN*TFA)yielding the two depicted taxol conjugates in a ration of 1:6 (carbonatevs carbamate, respectively).

[0342] 2. Analysis of Transport Across Skin

[0343] Varying concentrations (1 mM-100 μM) of taxol conjugated toeither biotinylated pentamer, heptamer, or nonamers of D-arginine (bior5, r7, or r9), dissolved in PBS, were applied to the back of nude mice.Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forthirty, sixty, and 120 minutes. At the end of this period animal wassacrificed, the relevant section of skin was excised, embedded inmounting medium (OCT) and frozen. Frozen sections were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.) as described inExample 1. Slides were analyzed by fluorescent microscopy.

[0344] Results

[0345] The conjugates of taxol with biotinylated heptamers and nonamersof D-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine and taxol, and nostaining was seen with a PBS control. The cytosol and nuclei of allcells in the field were fluorescent, indicating penetration into andthrough the epidermis and dermis. The staining pattern was consistentwith unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. These results demonstrate remarkable uptake onlywhen sufficient guanidinyl groups are included in the delivery-enhancingtransporter.

Example 12 Conjugate of Taxol and Delivery-Enhancing Transporter withpH-Releasable Linker

[0346] This Example demonstrates the use of a general strategy forsynthesizing prodrugs that have a delivery-enhancing transporter linkedto a drug by a linker that releases the drug from the delivery-enhancingtransporter upon exposure to physiological pH. In general, a suitablesite on the drug is derivatized to carry an α-chloroacetyl residue.Next, the chlorine is displaced with the thiol of a cysteine residuethat carries an unprotected amine. This scheme is shown in FIG. 16.

[0347] Methods

[0348] Synthesis of Taxol-2′-chloroacetyl

[0349] Taxol (89.5 mg, 104.9 μmol) was dissolved in CH₂Cl₂ (3.5 mL).Thesolution was cooled to 0° C. under an N₂-atmosphere. α-Chloroaceticanhydride (19.7 mg, 115.4 μmol) was added, followed by DIEA (14.8 mg,115.4 μmol). The solution was allowed to warm to room temperature. Afterthin layer chromatography (tlc) analysis indicated complete consumptionof starting material, the solvent was removed in vacuo and the crudematerial was purified by flash chromatography on silica gel (eluent:EtOAC/Hex 20%-50%) yielding the desired material (99.8 mg, quantitative)(FIG. 18).

[0350]¹H-NMR (CDCl₃): δ=8.13 (d, J=7.57 Hz, 2H), 7.72 (d, J=7.57 Hz,2H), 7.62-7.40 (m, 11H), 6.93 (d, J=9.14 Hz, 1H), 6.29-6.23 (m, 2H),6.01 (d, J=7.14 Hz, 1H), 5.66 (d, J=6.80 Hz, 1H), 5.55 (d, J=2.24 Hz,1H), 4.96 (d, J=8.79 Hz, 1H), 4.43 (m, 1H), 4.30 (d, J=8.29 Hz, 1H),4.20-4.15 (m, 2H), 3.81 (d, J=6.71 Hz, 1H), 2.56-2.34 (m, 3H), 2.45 (s,3H), 2.21 (s, 3H), 2.19 (m, 1H), 1.95-1.82 (m, 3H), 1.92 s, (3H), 1,67(s, 3H), 1.22 (s, 3H), 1.13 (s, 3H) ppm.

[0351]¹³C-NMR (CDCl₃): δ=203.6, 171.1, 169.7, 167.3, 167.0, 166.9,166.3, 142.3, 136.4, 133.6, 133.5, 132.9, 132.0, 130.1, 129.2, 121.1,128.7, 128.6, 127.0, 126.5, 84.3, 81.0, 79.0, 76.3, 75.4, 75.2, 75.0,72.2, 72.0, 58.4, 52.7, 45.5, 43.1, 40.1, 35.5, 26.7, 22.6, 22.0, 20.7,14.7, 9.5 ppm.

[0352] Linkage of Taxol to Delivery-Enhancing Transporter

[0353] The peptide (47.6 mg, 22.4 μmol) was dissolved in DMF (1.0 mL)under an N₂-atmosphere. DIEA (2.8 mg, 22.4 μmol) was added. A solutionof taxol-2′-chloroacetate (20.8 mg, 22.4 μmol) in DMF (1.0 mL) wasadded. Stirring at room temperature was continued for 6 hours. Watercontaining 0.1% TFA (1.0 mL) was added, the sample was frozen and thesolvents were lyophilized. The crude material was purified by RP-HPLC(eluent:water/MeCN*0.1%TFA: 85%-15%). A schematic of this reaction isshown in FIG. 18.

[0354] Synthesis of Related Conjugates

[0355] Using the conjugation conditions outlined above, the threeadditional conjugates shown in were synthesized.

[0356] Cytotoxicity Assay

[0357] The taxol conjugates were tested for cytotoxicity in a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) dyereduction. Results, which are shown in FIG. 20, demonstrate that thetaxol conjugated to r7 with a readily pH-releasable linker (CG 1062;R=Ac in the structure shown in FIG. 19) is significantly more cytotoxicthan either taxol alone or taxol conjugated to r7 with a less-readilypH-releasable linker (CG 1040; R=H in the structure shown in FIG. 19).

Example 13 Structure-Function Relationships of Fluorescently-LabeledPeptides Derived from Tat₄₉₋₅₇

[0358] Methods

[0359] General.

[0360] Rink amide resin and Boc₂O were purchased from Novabiochem.Diisopropylcarbodiimide, bromoacetic acid, fluorescein isothiocyanate(FITC-NCS), ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane,1,6-diaminohexane, trans-1,6-diaminocyclohexane, andpyrazole-1-carboxamidine were all purchased from Aldrich®. All solventsand other reagents were purchased from commercial sources and usedwithout further purification. The mono-Boc amines were synthesized fromthe commercially available diamines using a literature procedure (10equiv. of diamine and 1 equiv. of Boc₂O in chloroform followed by anaqueous work up to remove unreacted diamine) (34).

[0361] N-tert-butoxycarbonyl-1,6-trans-diaminocyclohexane.

[0362] Mp 159-161° C.; ¹H NMR (CDCl₃) δ 4.35 (br s, 1H), 3.37 (br s,1H), 2.61 (br s, 1H), 1.92-2.02 (m, 2H), 1.81-1.89 (m, 2H), 1.43 (s,9H), 1.07-1.24 (m, 4H) ppm; ¹³C NMR (D₆-DMSO) δ 154.9, 77.3, 49.7, 48.9,35.1, 31.4, 28.3 ppm; ES-MS (M+1) calcd 215.17, found 215.22.

[0363] General Procedure for Peptide Synthesis.

[0364] Tat₄₉₋₅₇ (RKKRRQRRR), truncated and alanine-substituted peptidesderived from Tat₄₉₋₅₇, Antennapedia₄₃₋₅₈ (RQIKIWFQNRRMKWKK), andhomopolymers of arginine (R5-R9) and d-arginine (r5-r9) were preparedwith an automated peptide synthesizer (ABI433) using standardsolid-phase Fmoc chemistry (35) with HATU as the peptide couplingreagent. The fluorescein moiety was attached via a aminohexanoic acidspacer by treating a resin-bound peptide (1.0 mmol) with fluoresceinisothiocyanate (1.0 mmol) and DIEA (5 mmol) in DMF (10 mL) for 12 h.Cleavage from the resin was achieved using 95:5 TFA/triisopropylsilane.Removal of the solvent in vacuo gave a crude oil which was trituratedwith cold ether. The crude mixture thus obtained was centrifuged, theether was removed by decantation, and the resulting orange solid waspurified by reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA). The productswere isolated by lyophilization and characterized by electrospray massspectrometry. Purity of the peptides was >95% as determined byanalytical reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA).

[0365] All peptides and peptoids synthesized contain an aminohexanoic(ahx) acid moiety attached to the N-terminal amino group with afluorescein moiety (Fl) covalently linked to the amino group of theaminohexanoic acid spacer. The carboxyl terminus of every peptide andpeptoid is a carboxamide.

[0366] Cellular Uptake Assay.

[0367] The arginine homopolymers and guanidine-substituted peptoids wereeach dissolved in PBS buffer (pH 7.2) and their concentration wasdetermined by absorption of fluorescein at 490 nm (ε=67,000). Theaccuracy of this method for determining concentration was established byweighing selected samples and dissolving them in a known amount of PBSbuffer. The concentrations determined by UV spectroscopy correlated withthe amounts weighed out manually. Jurkat cells (human T cell line),murine B cells (CH27), or human PBL cells were grown in 10% fetal calfserum and DMEM and each of these were used for cellular uptakeexperiments. Varying amounts of arginine and oligomers ofguanidine-substituted peptoids were added to approximately 3×10⁶ cellsin 2% FCS/PBS (combined total of 200 μL) and placed into microtiterplates (96 well) and incubated for varying amounts of time at 23° C. or4° C. The microtiter plates were centrifuged and the cells wereisolated, washed with cold PBS (3×250 μL), incubated with 0.05%trypsin/0.53 mM EDTA at 37° C. for 5 min, washed with cold PBS, andresuspended in PBS containing 0.1% propidium iodide. The cells wereanalyzed using fluorescent flow cytometry (FACScan, Becton Dickinson)and cells staining with propidium iodide were excluded from theanalysis. The data presented is the mean fluorescent signal for the 5000cells collected.

[0368] Inhibition of Cellular Uptake with Sodium Azide.

[0369] The assays were performed as previously described with theexception that the cells used were preincubated for 30 min with 0.5%sodium azide in 2% FCS/PBS buffer prior to the addition of fluorescentpeptides and the cells were washed with 0.5% sodium azide in PBS buffer.All of the cellular uptake assays were run in parallel in the presenceand absence of sodium azide.

[0370] Cellular Uptake Kinetics Assay.

[0371] The assays were performed as previously described except thecells were incubated for 0.5, 1, 2, and 4 min at 4° C. in triplicate in2% FCS/PBS (50 μl) in microtiter plates (96 well). The reactions werequenched by diluting the samples into 2% FCS/PBS (5 mL). The assays werethen worked up and analyzed by fluorescent flow cytometry as previouslydescribed.

[0372] Results

[0373] To determine the structural requirements for the cellular uptakeof short arginine-rich peptides, a series of fluorescently-labeledtruncated analogues of Tat₄₉₋₅₇ were synthesized using standardsolid-phase chemistry. See, e.g., Atherton, E. et al. SOLID-PHASEPEPTIDE SYNTHESIS (IRL: Oxford, Engl. 1989). A fluorescein moiety wasattached via an aminohexanoic acid spacer on the amino termini. Theability of these fluorescently labeled peptides to enter Jurkat cellswas then analyzed using fluorescent activated cell sorting (FACS). Thepeptide constructs tested were Tat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆(Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅ (Fl-ahx-RKKRRQR), Tat₅₀₋₅₇(Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇ (Fl-ahx-KRRQRRR). Differentiationbetween cell surface binding and internalization was accomplishedthroughout by running a parallel set of assays in the presence andabsence of sodium azide. Because sodium azide inhibits energy-dependentcellular uptake but not cell surface binding, the difference influorescence between the two assays provided the amount of fluorescenceresulting from internalization.

[0374] Deletion of one arginine residue from either the amine terminus(Tat₅₀₋₅₇) or the carboxyl terminus (Tat₄₉₋₅₆) resulted in an 80% lossof intracellular fluorescence compared to the parent sequence(Tat₄₉₋₅₇). From the one amino acid truncated analogs, further deletionof R-56 from the carboxyl terminus (Tat₄₉₋₅₅) resulted in an additional60% loss of intracellular fluorescence, while deletion of K-50 from theamine terminus (Tat₅₁₋₅₇) did not further diminish the amount ofinternalization. These results indicate that truncated analogs ofTat₄₉₋₅₇ are significantly less effective at the transcellular deliveryof fluorescein into Jurkat cells, and that the arginine residues appearto contribute more to cellular uptake than the lysine residues.

[0375] To determine the contribution of individual amino acid residuesto cellular uptake, analogs containing alanine substitutions at eachsite of Tat₄₉₋₅₇ were synthesized and assayed by FACS analysis (FIG.22). The following constructs were tested: A-49 (Fl-ahx-AKKRRQRRR), A-50(Fl-ahx-RAKRRQRRR), A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR),A-53 (Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55 20(Fl-ahx-RKKRRQARR), A-56 (Fl-ahx-RKKRRQRAR), and A-57(Fl-ahx-RKKRRQRRA). Substitution of the non-charged glutamine residue ofTat₄₉₋₅₇ with alanine (A-54) resulted in a modest decrease in cellularinternalization. On the other hand, alanine substitution of each of thecationic residues individually produced a 70-90% loss of cellularuptake. In these cases, the replacement of lysine (A-50, A-51) orarginine (A-49, A-52, A-55, A-56, A-57) with alanine had similar effectsin reducing uptake.

[0376] To determine whether the chirality of the transporter peptide wasimportant, the corresponding d-(d-Tat₄₉₋₅₇), retro-l-(Tat₅₇₋₄₉), andretro-inverso isomers (d-Tat₅₇₋₄₉) were synthesized and assayed by FACSanalysis (FIG. 23). Importantly, all three analogs were more effectiveat entering Jurkat cells then Tat₄₉₋₅₇. These results indicated that thechirality of the peptide backbone is not crucial for cellular uptake.Interestingly, the retro-l isomer (Tat57-49) which has three arginineresidues located at the amine terminus instead of one arginine and twolysines found in Tat₄₉₋₅₇ demonstrated enhanced cellular uptake. Thus,residues at the amine terminus appear to be important and that argininesare more effective than lysines for internalization. The improvedcellular uptake of the unnatural d-peptides is most likely due to theirincreased stability to proteolysis in 2% FCS (fetal calf serum) used inthe assays. When serum was excluded, the d- and l-peptides wereequivalent as expected.

[0377] These initial results indicated that arginine content isprimarily responsible for the cellular uptake of Tat₄₉₋₅₇. Furthermore,these results were consistent with our previous results where wedemonstrated that short oligomers of arginine were more effective atentering cells then the corresponding short oligomers of lysine,ornithine, and histidine. What had not been established was whetherarginine homo-oligomers are more effective than Tat₄₉₋₅₇. To addressthis point, Tat₄₉₋₅₇ was compared to the l-arginine (R5-R9) andd-arginine (r5-r9) oligomers. Although Tat49-57 contains eight cationicresidues, its cellular internalization was between that of R6 and R7(FIG. 24) demonstrating that the presence of six arginine residues isthe most important factor for cellular uptake. Significantly, conjugatescontaining 7-9 arginine residues exhibited better uptake than Tat₄₉₋₅₇.

[0378] To quantitatively compare the ability of these arginine oligomersand Tat₄₉₋₅₇ to enter cells, Michaelis-Menton kinetic analyses wereperformed. The rates of cellular uptake were determined after incubation(3° C.) of the peptides in Jurkat cells for 30, 60, 120, and 240 seconds(Table 1). The resultant K_(m) values revealed that r9 and R9 enteredcells at rates approximately 100-fold and 20-fold faster than Tat₄₇₋₅₉respectively. For comparison, Antennapedia₄₃₋₅₈ was also analyzed andwas shown to enter cells approximately 2-fold faster than Tat₄₇₋₅₉, butsignificantly slower than r9 or R9. TABLE 1 Michaelis-Menton kinetics:Antennapedia₄₃₋₅₈ (Fl-ahx- RQIKIWFQNRRMKWKK). peptide K_(m) (μM) V_(max)Tat₄₉₋₅₇ 770 0.38 Antennapedia₄₃₋₅₈ 427 0.41 R9 44 0.37 r9 7.6 0.38

Example 14 Design and Synthesis of Peptidomimetic Analogs of Tat₄₉₋₅₇

[0379] Methods

[0380] General Procedure for Peptoid Polyamine Synthesis.

[0381] Peptoids were synthesized manually using a fritted glassapparatus and positive nitrogen pressure for mixing the resin followingthe literature procedure developed by Zuckermann. See, e.g., Murphy, J.E. et al., Proc. Natl. Acad. Sci. USA 95, 1517-1522 (1998); Simon, R. J.et al., Proc. Natl. Acad. Sci. USA 89, 9367-9371 (1992); Zuckermann, R.N. et al., J. Am. Chem. Soc. 114, 10646-10647 (1992). Treatment ofFmoc-substituted Rink amide resin (0.2 mmol) with 20% piperidine/DMF (5mL) for 30 min (2×) gave the free resin-bound amine which was washedwith DMF (3×5 mL). The resin was treated with a solution of bromoaceticacid (2.0 mmol) in DMF (5 mL) for 30 min. This procedure was repeated.The resin was then washed (3×5 mL DMF) and treated with a solution ofmono-Boc diamine (8.0 mmol) in DMF (5 mL) for 12 hrs. These two stepswere repeated until an oligomer of the required length was obtained(Note: the solution of mono-Boc diamine in DMF could be recycled withoutappreciable loss of yield). The resin was then treated withN-Fmoc-aminohexanoic acid (2.0 mmol) and DIC (2.0 mmol) in DMF for 1 hand this was repeated. The Fmoc was then removed by treatment with 20%piperidine/DMF (5 mL) for 30 min. This step was repeated and the resinwas washed with DMF (3×5 mL). The free amine resin was then treated withfluorescein isothiocyanate (0.2 mmol) and DIEA (2.0 mmol) in DMF (5 mL)for 12 hrs. The resin was then washed with DMF (3×5 mL) anddichloromethane (5×5 mL). Cleavage from the resin was achieved using95:5 TFA/triisopropylsilane (8 mL). Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether (20 mL). The crudemixture thus obtained was centrifuged, the ether was removed bydecantation, and the resulting orange solid was purified byreverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA). The products were isolatedby lyophilization and characterized by electrospray mass spectrometryand in selected cases by ¹H NMR spectroscopy.

[0382] General Procedure for Perguanidinylation of Peptoid Polyamines.

[0383] A solution of peptoid amine (0.1 mrnol) dissolved in deionizedwater (5 mL) was treated with sodium carbonate (5 equivalents per amineresidue) and pyrazole-l-carboxamidine (5 equivalents per amine residue)and heated to 50° C. for 24-48 hr. The crude mixture was then acidifiedwith TFA (0.5 mL) and directly purified by reverse-phase HPLC (H₂O/CH₃CNin 0.1% TFA). The products were characterized by electrospray massspectrometry and isolated by lyophilization and firther purified byreverse-phase HPLC. The purity of the guanidine-substituted peptoidswas >95% as determined by analytical reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA).

[0384] Results

[0385] Utilizing the structure-function relationships that had beendetermined for the cellular uptake of Tat₄₇₋₅₉, we designed a set ofpolyguanidine peptoid derivatives that preserve the 1,4 backbone spacingof side chains of arginine oligomers, but have an oligo-glycine backbonedevoid of stereogenic centers. These peptoids incorporatingarginine-like side chains on the amide nitrogen were selected because oftheir expected resistance to proteolysis, and potential ease andsignificantly lower cost of synthesis (Simon et al., Proc. Natl. Acad.Sci. USA 89:9367-9371 (1992); Zuckermann, et al., J. Am. Chem. Soc.114:10646-10647 (1992). Furthermore, racemization, frequentlyencountered in peptide synthesis, is not a problem in peptoid synthesis;and the “sub-monomer” peptoid approach allows for facile modification ofside-chain spacers. Although the preparation of an oligurea andpeptoid-peptide hybrid (Hamy, et al, Proc. Natl. Acad. Sci. USA94:3548-3553 (1997)) derivatives of Tat₄₉₋₅₇ have been previouslyreported, their cellular uptake was not explicitly studied.

[0386] The desired peptoids were prepared using the “sub-monomer”approach (Simon et al.; Zuckermann et al.) to peptoids followed byattachment of a fluorescein moiety via an aminohexanoic acid spacer ontothe amine ternini. After cleavage from the solid-phase resin, thefluorescently labeled polyamine peptoids thus obtained were converted ingood yields (60-70%) into polyguanidine peptoids by treatment withexcess pyrazole-1-carboxamidine (Bernatowicz, et al., J. Org. Chem.57:2497-2502 (1992) and sodium carbonate (as shown in FIG. 25).Previously reported syntheses of peptoids containing isolated N-Argunits have relied on the synthesis of N-Arg monomers (5-7 steps) priorto peptoid synthesis and the use of specialized and expensive guanidineprotecting groups (Pmc, Pbf) (Kruijtzer, et al., Chem. Eur. J.4:1570-1580 (1998); Heizmann, et al. Peptide Res. 7:328-332 (1994). Thecompounds reported here represent the first examples ofpolyguanidinylated peptoids prepared using a perguanidinylation step.This method provides easy access to polyguanidinylated compounds fromthe corresponding polyamines and is especially useful for the synthesisof perguanidinylated homooligomers. Furthermore, it eliminates the useof expensive protecting groups (Pbf, Pmc). An additional example of aperguanidinylation of a peptide substrate using a noveltriflyl-substituted guanylating agent has recently been reported(Feichtinger, et al., J. Org. Chem. 63:8432-8439 (1998)).

[0387] The cellular uptake of fluorescently labeled polyguanidineN-arg5,7,9 peptoids was compared to the corresponding d-argininepeptides r5,7,9 (similar proteolytic properties) using Jurkat cells andFACS analysis. The amount of fluorescence measured inside the cells withN-arg5,7,9 was proportional to the number of guanidine residues:N-arg9 >N-arg7 >N-arg5 (FIG. 26), analogous to that found for r5,7,9.Furthermore, the N-arg5,7,9 peptoids showed only a slightly lower amountof cellular entry compared to the corresponding peptides, r5,7,9. Theresults demonstrate that the hydrogen bonding along the peptide backboneof Tat₄₉₋₅₇ or arginine oligomers is not a required structural elementfor cellular uptake and oligomeric guanidine-substituted peptoids can beutilized in place of arginine-rich peptides as molecular transporters.The addition of sodium azide inhibited internalization demonstratingthat the cellular uptake of peptoids was also energy dependent.

Example 15 The effect of Side Chain Length on Cellular Uptake

[0388] After establishing that the N-arg peptoids efficiently crossedcellular membranes, the effect of side chain length (number ofmethylenes) on cellular uptake was investigated. For a given number ofguanidine residues (5,7,9), cellular uptake was proportional to sidechain length. Peptoids with longer side chains exhibited more efficientcellular uptake. A nine-mer peptoid analog with a six-methylene spacerbetween the guanidine head groups and the backbone (N-hxg9) exhibitedremarkably higher cellular uptake than the corresponding d-arginineoligomer (r9). The relative order of uptake was N-hxg9 (6methylene)>N-btg9 (4 methylene)>r9 (3 methylene)>N-arg9 (3methylene)>N-etg9 (2 methylene) (FIG. 27). Of note, the N-hxg peptoidsshowed remarkably high cellular uptake, even greater than thecorresponding d-arginine oligomers. The cellular uptake of thecorresponding heptamers and pentamers also showed the same relativetrend. The longer side chains embodied in the N-hxg peptoids improvedthe cellular uptake to such an extent that the amount of internalizationwas comparable to the corresponding d-arginine oligomer containing onemore guanidine residue (FIG. 28). For example, the N-hxg7 peptoid showedcomparable cellular uptake to r8.

[0389] To address whether the increase in cellular uptake was due to theincreased length of the side chains or due to their hydrophobic nature,a set of peptoids was synthesized containing cyclohexyl side chains.These are referred to as the N-chg5,7,9 peptoids. These contain the samenumber of side chain carbons as the N-hxg peptoids but possess differentdegrees of freedom. Interestingly, the N-chg peptoid showed much lowercellular uptake activity than all of the previously assayed peptoids,including the N-etg peptoids (FIG. 29). Therefore, the conformationalflexibility and sterically unencumbered nature of the straight chainalkyl spacing groups is important for efficient cellular uptake.

[0390] Discussion

[0391] The nona-peptide, Tat₄₉₋₅₇, has been previously shown toefficiently translocate through plasma membranes. The goal of thisresearch was to determine the structural basis for this effect and usethis information to develop simpler and more effective moleculartransporters. Toward this end, truncated and alanine substitutedderivatives of Tat₄₉₋₅₇ conjugated to a fluoroscein label was prepared.These derivatives exhibited greatly diminished cellular uptake comparedto Tat₄₉₋₅₇, indicating that all of the cationic residues of Tat₄₉₋₅₇are required for efficient cellular uptake. When compared with ourprevious studies on short oligomers of cationic oligomers, thesefindings suggested that an oligomer of arginine might be superior toTat₄₉₋₅₇ and certainly more easily and cost effectively prepared.Comparison of short arginine oligomers with Tat₄₉₋₅₇ showed that membersof the former were indeed more efficiently taken into cells. This wasfurther quantified for the first time bt Michaelis-Menton kineticsanalysis which showed that the R9 and r9 oligomers had Km values 30-foldand 100-fold greater than that found for Tat₄₉₋₅₇.

[0392] Given the importance of the guanidino head group and the apparentinsensitivity of the oligomer chirality revealed in our peptide studies,we designed and synthesized a novel series of polyguanidine peptoids.The peptoids N-arg5,7,9, incorporating the arginine side chain,exhibited comparable cellular uptake to the corresponding d-argininepeptides r5,7,9, indicating that the hydrogen bonding along the peptidebackbone and backbone chirality are not essential for cellular uptake.This observation is consistent with molecular models of these peptoids,arginine oligomers, and Tat₄₉₋₅₇, all of which have a deeply embeddedbackbone and a guanidinium dominated surface. Molecular models furtherreveal that these structural characteristics are retained in varyingdegree in oligomers with different alkyl spacers between the peptoidbackbone and guanidino head groups. Accordingly, a series of peptoidsincorporating 2-(N-etg), 4-(N-btg), and 6-atom (N-hxg) spacers betweenthe backbone and side chain were prepared and compared for cellularuptake with the N-arg peptoids (3-atom spacers) and d-arginineoligomers. The length of the side chains had a dramatic affect oncellular entry. The amount of cellular uptake was proportional to thelength of the side chain with N-hxg>N-btg>N-arg>N-etg. Cellular uptakewas improved when the number of alkyl spacer units between the guanidinehead group and the backbone was increased. Significantly, N-hxg9 wassuperior to r9, the latter being 100-fold better than Tat₄₉₋₅₇. Thisresult led us to prepare peptoid derivatives containing longer octylspacers (N-ocg) between the guanidino groups and the backbone. Issuesrelated to solubility prevented us from testing these compounds.

[0393] Because both perguanidinylated peptides and perguanidinylatedpeptoids efficiently enter cells, the guanidine head group (independentof backbone) is apparently the critical structural determinant ofcellular uptake. However, the presence of several (over six) guanidinemoieties on a molecular scaffold is not sufficient for active transportinto cells as the N-chg peptoids did not efficiently translocate intocells. Thus, in addition to the importance of the guanidine head group,there are structure/conformational requirements that are significant forcellular uptake.

[0394] In summary, this investigation identified a series of structuralcharacteristics including sequence length, amino acid composition, andchirality that influence the ability of Tat₄₉₋₅₇ to enter cells. Thesecharacteristics provided the blueprint for the design of a series ofnovel peptoids, of which 17 members were synthesized and assayed forcellular uptake. Significantly, the N-hxg9 transporter was found to besuperior in cell uptake to r9 which was comparable to N-btg9. Hence,these peptoid transporters proved to be substantially better thanTat₄₉₋₅₇. This research established that the peptide backbone andhydrogen bonding along that backbone are not required for cellularuptake, that the guanidino head group is superior to other cationicsubunits, and most significantly, that an extension of the alkyl chainbetween the backbone and the head group provides superior transporters.In addition to better uptake performance, these novel peptoids offerseveral advantages over Tat₄₉₋₅₇ including cost-effectiveness, ease ofsynthesis of analogs, and protease stability. These features along withtheir significant water solubility (>100 mg/mL) indicate that thesenovel peptoids could serve as effective transporters for the moleculardelivery of drugs, drug candidates, and other agents into cells.

Example 16 Synthesis of Itraconazole-Transporter Conjugate

[0395] This Example provides one application of a general strategy forattaching a delivery-enhancing transporter to a compound that includes atriazole structure. The scheme, using attachment of itraconazole to anarginine (r7) delivery-enhancing transporter as an example, is shown inFIG. 30. In the scheme, R is H or alkyl, n is 1 or 2, and X is ahalogen.

[0396] The reaction involves making use of quaternization of a nitrogenin the triazole ring to attach an acyl group that has a halogen (e.g.,Br, Fl, I) or a methyl ester. Compound 3 was isolated by HPLC. ProtonNMR in D₂O revealed itraconazole and transporter peaks.

[0397] The methyl ester provided yields of 70% and greater, while yieldsobtained using the Br-propionic acid/ester pair were 40-50%. The acylderivative is then reacted with the amine of the delivery-enhancingtransporter to form the conjugate. Alternatively, the halogenated acylgroup can first be attached to the transporter molecule through an amidelinkage, after which the reaction with the drug compound is conducted.

Example 17 Preparation of FK506 Conjugates

[0398] This Example describes the preparation of conjugates in whichFK506 is attached to a delivery-enhancing transporter. Two differentlinkers were used, each of which released FK506 at physiological pH (pH5.5 to 7.5), but had longer half-lives at more acidic pH. These schemesare diagrammed in FIGS. 31A and B.

[0399] Linker 1: 6-maleimidocaproic Hydrazide Trifluroacetate (Scheme Iand II)

[0400] A solution of FK506 (1) (0.1 g, 124.4 μmol), 6-maleiimidocaproichydrazide trifluoroacetate (2) (0.126 g, 373.2 μmol) and trifluoroaceticacid (catalytic, 1 μL) in anhydrous methanol (5 mL) was stirred at roomtemperature for 36 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system—dichloromethane (95): methanol (5),R_(f)=0.3]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (96): methanol (4) as eluent togive the hydrazone 3 (0.116 g, 92%).

[0401] A solution of the above hydrazone (3) (0.025 g, 24.7 μmol),transporter (1×, Bacar₉CCONH₂.9TFA, Bacar₇CCONH₂.7TFA, BacaCCONH₂,NH₂r₇CCONH₂.8TFA, NH₂R₇CCONH₂.8TFA) and diisopropylethylamine (1×) inanhydrous dimethylformamide (1 mL) were stirred under nitrogen at roomtemperature for 36 h when TLC indicated the complete disappearance ofthe starting hydrazone. Solvent was evaporated from the reaction mixtureand the residue purified by reverse phase HPLC using trifluoroaceticacid buffered water and acetonitrile.

[0402] Yields of conjugates with various transporters:

[0403] Conjugate with Bacar₉CCONH₂.9TFA (4)—73%

[0404] Bacar₇CCONH₂.7TFA (5)—50%

[0405] BacaCCONH₂ (6)—52.9%

[0406] NH₂r₇CCONH₂.8TFA (7)—43.8%

[0407] NH₂R₇CCONH₂.8TFA (8)—62.8%

[0408] Structures of all the products were confirmed by 1H-NMR spectraand TOF MS analysis.

[0409] Linker 2: 2-(2-pyridinyldithio)ethyl Hydrazine Carboxylate(Scheme III and IV)

[0410] A solution of FK506 (1) (0.1 g, 124.4 μmol),2-(2-pyridinyldithio) ethyl hydrazine carboxylate (9) (0.091 g, 373.2μmol) and trifluoroacetic acid (catalytic, 1 μL) in anhydrous methanol(5 mL) was stirred at room temperature for 16 h. The reaction wasmonitored by thin layer chromatography that showed almost completedisappearance of the starting material. [TLC solvent system—ethylacetate R_(f)=0.5]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (97): methanol (3) as eluent togive the hydrazone 10 (0.091 g, 71%)

Example 18 Differential Uptake of Transporters in the GastrointestinalTract Methods

[0411] Gastrointestinal Absorption Protocol

[0412] Experiments were performed on 8- to 10-week-old female SwissWebster mice purchased from Taconic (Germantown, N.Y.). Mice wereanesthetized with Nembutal and a midline incision was made along theabdomen. Intestines were measured, tied off at both ends of the desiredsection with sutures, and biotinylated peptides were injected into thelumen (approximately 100λ/inch). After a fifteen minute incubation, thetissue was removed and the lumen was gently washed with PBS.

[0413] To determine whether CellGate transporters could enter thesquamous epithelia of the oral cavity, mice were anesthetized withNembutal, their heads tipped to one side and solutions of thebiotinylated peptides were placed in their mouths. After fifteen minutesthe liquid was removed by pipette.

[0414] Preparation of Histological Sections of Regions of theGastrointestinal Tract

[0415] Immediately following incubation with biotinylated peptides, theanesthetized rodents were sacrificed by cervical dislocation and thetied off sections of the GI tract were removed. The lumens of thevarious sections were filled with OCT using a plastic tipped syringe,immersed in OCT filled boats, and snap frozen in a 2-methyl-butane/dryice solution. Frozen sections were allowed to warm slightly and 2 mmthick sagital cuts were made using a steel razor blade. The cuts wereplaced into OCT molds and snap frozen in a 2-methyl-butane/dry icesolution. Sections (5 μm) were cut on the cryostat, fixed in acetone at4° C. for 10 minute, and allowed to air dry. After rehydration in PBSfor 5 minutes, sections were blocked with normal horse serum, washed,incubated with streptavidin-FITC (20 μg/ml) for 30 minutes, washed, andmounted with mounting medium containing propidium iodide (PI).

[0416] Tissue Culture

[0417] Caco-2 cells were acquired from ATCC, thawed, and grown in DMEMcontaining penicillin, streptomycin, glutamine, and 10% fetal bovineserum for 3-5 days to confluency (>250,000 cells/cm²) and then passagedat 60,000 cells/cm² for several passage cycles. Passage numbers 25-29were plated on Snapwell 12 mm diameter 0.4-micron pore sizepolycarbonate membranes (Corning Costar, Corning, N.Y.) at 60,000 cellsper membrane and allowed to grow for at least 21 days, with mediachanged every other day.

[0418] Cellular Uptake in Caco-2 Cells

[0419] To analyze the penetration of fluorescent oligomers of D-arginineinto the cells when in suspension, the monolayers were treated withtrypsin (4.5 ml of a 0.05% solution Gibco, Grand Rapids, Mich.) and theindividual cell suspension was spun down. Cells were resuspended,counted and treated with varying concentrations of Fl aca r5, Fl aca r7,Fl aca r9 and Fl aca k9 (from 50-0.8 μM) for five minutes, washed,resuspended in 400λ PBS, 2%FBS, 40ngPI/ml and analyzed by flowcytometry.

[0420] To analyze the ability of the fluorescent peptides to enterCaco-2 cells when part of a monolayer, the cells were seeded in lab-tekflaskette microscope slides (Nalge Nunc Int., Naperville, Ill.) in 4 mlat a density of 60,000 cells/ml and grown for 21 days with the mediabeing changed every other day. Once the monolayer was established, itwas incubated with 100 μM Fl aca r9 CONH2 for a five minutes. Themonolayer was subsequently washed with PBS/2% FBS twice to removelabeled peptide and analyzed using fluorescent microscopy.

[0421] Transport Across Monolayers of Caco-2 Cells

[0422] The experiments analyzing whether short oligomers of D-argininecould cross monolayers of Caco-2 cells were performed using a voltageclamp amplifier and Easymount side-by-side horizontal diffusion chamber(Physiologic Instruments, San Diego, Calif.) with a 95% oxygen 5% carbondioxide gas lift system connected to a recirculating water bath.

[0423] Current measurements (I₂-I₁) were taken at five minute intervals,including 10 minutes prior to the addition of the test compounds toinsure monolayers were intact. At zero time test compounds were added atthe proper concentrations and measurements recorded. Measurements weretaken for 60 minutes at 5 minute intervals; transepithelia electricalresistance (TEER) and short circuit current (Isc) were subsequentlycalculated from these values.

[0424] Synthetic Chemistry

[0425] Cyclosporin A

[0426] The details of the CsA conjugate used in this study have beendescribed previously (Rothbard et al. Nature Medicine 6, 1253 2000). Seealso, FIG. 6. Briefly, CsA was conjugated to a heptamer of D-argininethrough a pH sensitive linker as shown in FIG. 6A. The resultantconjugate is stable at acidic pH but at pH>7 it undergoes anintramolecular cyclization involving addition of the free amine to thecarbonyl adjacent to CsA (FIG. 6B), which results in the release ofunmodified CsA.

[0427] Taxol

[0428] Taxol was treated with α-chloro acetic anhydride delivering theC-2′ chloro acetyl derivative 12 in essentially quantitative yield.

[0429] The halogen was displaced by the thiol of the N-terminal (L)cysteine containing heptamer of D-arginine. Conjugations were performedat room temperature in DMF in the presence of DIEA. The final productswere isolated by RP-HPLC and lyophilized to yield TFA salts, which werevery hygroscopic and readily dissolve in water.

[0430] Compound 13 was designed to release taxol via a nucleophilicattack of the N-terminal nitrogen onto the C2′ ester carbonyl. Theprotonation state of this nitrogen is crucial for this mechanism, sinceonly the free amine will be capable of this release. Additionally, bothconjugates share a common α-hetero atom substituted acetate moietymaking them susceptible to simple ester hydrolysis. This offers anadditional release pathway.

[0431] Intracolonic Injections

[0432] Wistar rats, females of approximately 200-300g (Simonsen, Gilroy,Calif.), were anesthetized, their abdomens were shaved, midlineincisions were made, and a one-half inch section of the ascending colonin each animal was tied off with sutures. Taxol and cyclosporin (5mg/kg) were injected as solutions in a 1:1 v/v Cremophor EL:ethanolmixture, whereas equivalent molar amounts of the r7 conjugates wereinjected in PBS. In all cases, the volume injected was approximately500λ. The colon was placed back into the cavity, and the incision wasclosed using sutures.

[0433] Blood samples were taken from the tail vein at time zero (priorto drug injection), and every thirty minutes for the duration of theexperiment, which was empirically determined, with the exception of oneanimal that expired after ninety minutes. Clotting was inhibited bytransferring the blood to glass tubes containing 100λ of 0.5% EDTA, andthe blood was frozen.

[0434] Drug Extraction from Whole Blood and HPLC MS MS Analysis

[0435] Either taxol or cyclosporin was extracted from the whole bloodusing a modification of literature procedures. Briefly, whole blood(100λ) was transferred to a screw capped glass tube containing five mlsof diethyl ether. The sample was vortexed vigorously for two minutes,centrifuged, and frozen in dry ice/methanol. The ethereal layer wastransferred to another glass tube and the ether was evaporated. In thecase of cyclosporin, the residue was resuspended in 1.5 mls of methanol,water, acetonitrile (3:2:1), while for taxol the residue was resuspendedin 1.5 mls methanol:acetonitrile (1:1). Samples were placed into aPerkin-Elmer series 200 autosampler and sequencially injected onto a C₁₈reverse column at 70° C. connected to a Shimadzu HPLC system, elutedwith 70% methanol, 30% aqueous ammonium formate buffer, and the effluentwas analyzed on a PE Sciex API 3000 tandem mass spectrometer. Knownamounts of either cyclosporin A (10-1000 ng/ml) or taxol (1-1000 ng/ml)were added to whole blood and extracted as previously described togenerate standard curves. Cyclosporin A was monitored by the twotransitions from both 1220 to 1203 daltons and 1220 to 100 daltons. The1220 species corresponds to the cyclosporin A+ammonia, the 1203 isprotonated cyclosporin, while 100 is a known fragment. Taxol wasmonitored by the two transitions from both 872 to 855 daltons and 872 to110 daltons. The 872 species corresponds to the ammonium adduct oftaxol, the 855 is the protonated parent compound, whereas 110 is thepredominant fragment seen in the second quadrupole.

[0436] The total amount of cyclosporin A and taxol in the samples wasdetermined by comparing the integrated area of the appropriate peak withvalues established by the standard curves.

Results

[0437] Differential Uptake of Transporters in the Gastrointestinal Tract

[0438] Short oligomers of D-arginine have been shown to cross rapidlyand efficiently the plasma membrane of a large variety of cell linesgrown in suspension. In addition, they have been shown to penetratemultiple layers of the skin when applied topically, multiple layers ofendothelial and smooth muscle cells of veins and arteries when injectedintravenously, and multiple cell layers of lung tissue when inhaled. Todetermine whether these compounds could enter the nonkeratinizedepithelia of the gastrointestinal tract, sections of the small and largeintestines of fasted mice were tied off and solutions of biotinylatednonamers of D-arginine, bio aca r9 CONH₂, (100 μM) were injected. Afterfifteen minutes of exposure, the relevant section of tissue wasdissected, frozen, sectioned, and stained with Streptavidin fluoresceinto define the location of the biotinylated peptide, and propidium iodideto counterstain all the nuclei in the section.

[0439] When injected into the lumen of murine duodenum no detectablestaining over background was observed. Poor staining also was seen whenthe biotinylated peptide was injected into the jejunum. If multiplesections were scanned detectable staining was seen on the tips of somevilli. The first sign of uniform staining was observed when sections ofthe ileum were analyzed. The staining was localized to the tips of themicrovilli and did not extend into the crypt cells. Although seenthroughout all sections of the ileum, the observed fluorescence did notapproach the level of intensity previously observed in the skin, lungs,or the endothelial cells of arteries or veins.

[0440] The relatively poor staining of the small intestine markedlydiffered from that seen when regions of the colon were examined. In boththe ascending and transverse sections of the large intestine,biotinylated nonamers of arginine stained all surface areas of thevilli, and penetrated several cell layers, reminiscent of the intensestaining of the epidermis and dermis when applied topically. Inaddition, the crypt cells were heavily stained in the colonic sectionsand evidence for penetration of the full thickness of the section wasobserved in several areas.

[0441] The histological analysis demonstrated that the uptake of bio r9into the nonkeratinized epithelia layers of the GI tract variedsignificantly, with uptake increasing with distance from the gastricpylorus. The staining observed in the ileum was greater than that seenin the jejunum, which was greater than the duodenum. The greateststaining was seen in the ascending and transverse regions of the colon.Without intending to limit the invention to a particular theory ormechanism, one theory is that the composition and amount of mucus liningthe epithelia might be an important factor in the differential staining.

[0442] Cellular Uptake into Caco-2 Cells

[0443] When Caco-2 cells, commercially-available human colon cells, wereincubated in suspension with varying amounts of fluorescently labeledpentamers, heptamers, and nonamers of D-arginine (Fl aca r5, Fl aca r7,Fl aca r9) or nonamers of lysine (Fl aca k7), and analyzed by flowcytometry, a pattern similar to that previously seen in a variety ofother suspension cells (Mitchell et al. Peptide Research 56, 318 (2000))was observed (FIG. 32). Uptake of the fluorescent peptides increasedwith arginine content with r9 being more effective than r7, which wasmore effective than r5. All the polymers of arginine entered cells moreeffectively than the nonamers of lysine. Both the rate, the relativeamount of fluorescence, and the lack of apparent efflux of theinternalized fluorescent peptides over an extended period of time(several hours) were reminiscent of earlier experiments withlymphocytes.

[0444] To confirm that the rapid penetration of the short oligomers ofarginine into Caco-2 cells was not an artifact seen only when the cellswere in suspension, Fl aca r9 CONH₂ (50 μM) was incubated for fiveminutes with Caco-2 cells grown as a monolayer on a microscope slide,washed, and analyzed by fluorescent microscopy. Consistent with the flowcytometry analysis of the suspension cells, virtually every cell in themonolayer was fluorescent after five minutes.

[0445] The ability of the peptides to rapidly enter Caco-2 cells wasfirmly established by placing a monolayer of Caco-2 cells as a membranein a diffusion chamber and exposing it to fluorescent transporter drugconjugates between taxol and oligomers of arginine of different length.Varying concentrations of the taxol conjugates (50-0.08 μM) were addedto the apical side and exposed to the monolayer of Caco-2 cells forthree minutes. The membrane was removed from the apparatus, the cellstrypsinized, and the resulting cell suspension was analyzed by flowcytometry (FIG. 33). As with the peptides alone, the drug conjugatesquickly and efficiently entered the cells composing the monolayer withthose containing more arginine subunits being more effective.

[0446] Without intending to limit the theory of the invention, theseexperiments provide strong support for the hypothesis that shortoligomers of arginine, either conjugated to fluorescein or therapeuticdrugs, such as taxol, rapidly enter Caco-2 cells both in suspension andwhen grown in monolayers.

[0447] Transport Across Caco-2 Monolayers

[0448] Crossing the luminal membrane of the gut epithelia is necessaryto increase blood levels of a delivered (e.g., buccal administered)drug. To determine whether the transporters could cross the gutepithelia, monolayers of Caco-2 cells were grown in culture and placedas a membrane in a commercially available diffusion chamber. Theintegrity of the membranes was established by demonstrating that thetransepithelial electronic resistance (TEER) was always greater than 100ohm cm² (FIG. 34). Such a pattern of stable resistance only is observedwhen the membrane is intact with no significant spaces between thecells.

[0449] Additional evidence that the membrane was both viable andcontiguous was that Lucifer Yellow (200 μM) was not transported acrossthe monolayer, whereas hydrocortisone was transported at amountsconsistent with published reports (FIG. 35).

[0450] When a variety of fluorescent oligomers of D- or L-arginine,ranging from four to 15 subunits, were placed in the apical chamber inmultiple experiments with a large number of membranes, none weresignificantly transported into the basolateral chamber (FIG. 35).

[0451] Taken together, the data presented herein are consistent with themodel that short oligomers of arginine either conjugated to deliveredcompounds such as fluorescein or taxol rapidly enter but are nottransported across Caco-2 cell monolayers. This model also implies thatthe transporters of the invention do not enter Caco-2 cells byendosomes, which are transported across colonic epithelium and excretedon the basolateral side by a well understood pathway. CellGatetransporters appear to enter the cytosol directly and do not have a highrate of efflux on the basolateral side.

[0452] Measurement of Drug Blood Levels after Intracolonic Injections

[0453] Although short oligomers of arginine labeled with fluorescein,either alone, or when conjugated to either taxol or cyclosporin, wereunable to cross a monolayer of Caco-2 cells in vitro, the cell line maynot precisely mimic the in vivo behavior of the colon. Furthermore, thepattern of fluorescence seen in colon tissue after incubation with shortoligomers of arginine demonstrated that the peptides penetrated intolayers known to be vascularized.

[0454] To determine whether the transporters of the invention couldenhance the delivery of orally administered drugs, releasable conjugatesof cyclosporin A and taxol were injected intracolonically and theresulting blood levels of the released drugs were measured by LC MS MS.

[0455] The first experiment was designed to measure blood levels of CsAafter intracolonic injection of 5 mg/kg of CsA in Cremophor EL:ethanolcompared with an equimolar amount of a releasable r7 conjugate of CsAdissolved in phosphate buffered saline. In the case of the parent drug,blood levels rose to approximately 25 ng/ml of CsA after thirty minutes,and then rapidly fell off to levels close to baseline levels (FIG. 36).No further data was obtained because after 90 minutes the animal died.In contrast, when an equimolar amount of CsA-r7 conjugate was injected,detectable levels of CsA in the blood were observed only after 3 hours(FIG. 36). To determine whether the altered pharmacokinetics of CsA whenconjugated to a short oligomer of arginine was reproducible, a third ratwas injected with 10 mg/kg equivalent of the water soluble, releasableconjugate.

[0456] The rate of uptake of CsA in the blood of this animal resembledthe animal injected with 5 mg/kg of the conjugate. With more conjugateadministered a small increase was seen at 30 minutes, but larger amountsappeared in the blood only after two hours, with blood levelsapproaching 45 ng/ml after three hours. The overall pattern was similarin the two animals injected with the conjugate. In both cases theoverall amount of CsA measured in the circulation was significantlygreater than observed when CsA was injected.

[0457] The half-life of the CsA conjugate was approximately ninetyminutes, which was consistent with the delay in the appearance of CsA inthe blood relative to the parent compound. This fact combined with thehistological data demonstrating rapid and efficient uptake in thecolumnar epithelium of the colon and the failure of nonreleasableconjugates of CsA or taxol to cross monolayers of colonic cells invitro, leads to a sensible and simple model describing the phenomena.Without intending to limit the invention to a particular mechanism ortheory, it appears that the conjugates enter the columnar epithelia ofthe colon with greater efficiency and more rapidly than CsA injected inCremophor, but were retained in the epithelial cells and did not crossthe endothelial cells surrounding the capillaries until the conjugatehydrolyzed. Once released, the CsA freely difflised, or was activelytransported, into the blood.

[0458] To test this hypothesis, taxol and two different r7-taxolconjugates were injected into the colon and blood levels of the drugwere measured at thirty minute intervals. The two r7-taxol conjugateshad significantly different half-lives (10 minutes and 5 hours) and wereused to test the hypothesis that the hydrolysis of the drug conjugatewas the rate limiting step in appearance of the drug in the circulation.If true, the premise predicted that the r7-taxol conjugate with the tenminute half life would release taxol in the epithelia so that it couldbe detected in the circulation at the thirty minute time point. Incontrast, taxol should not be released from the more stable r7 conjugate(t_(½)=5 hours) and should not be detected in the blood samples takenduring the experiment.

[0459] This premise was supported by the appearance of taxol in theblood (FIG. 37). Detectable levels of taxol did not appear in the bloodwhen injected in the colon until 2.5 hours with subsequent waves at 4and 5.5 hours. The oscillating levels of taxol in the blood as afunction of time were consistent with published studies with the patternbeing rationalized to the ability of Cremophor to sequester some of thematerial and act as a timed release vehicle. In contrast, when a labile,water soluble r7 conjugate of taxol (13) was administered, greater than200 ng/ml of taxol was observed at the earliest time point (30 minutes)which continued to increase up to 1 hour, at which point the levelsslowly diminished out to five hours. As predicted, injection of the morestable, water soluble r7 conjugate of taxol (14) did not result insignificant blood levels of taxol within five hours. These data supportthe hypothesis that transporters of the invention enter, but do nottransverse the colonic epithelium. They can improve oral bioavailabilityof drugs both by dramatically improving water solubility and byincreasing the rate of uptake of the conjugate in the colon. Ultimatedelivery of the drug into the blood stream appears to depend on thedrug's inherent ability to diffuse through biological membranes and therate of release from the conjugate.

Discussion

[0460] A significant discovery in the experiments described herein isthe failure of the peptides to enter the epithelia of the duodenum. Thisrepresents the first example of the transporters not entering a celltype or tissue. The pattern of increasing uptake the further the tissueextended from the gastric pylorus was intriguing, most likely due to agradient of an inhibitor, such as a negatively charged mucus. Thefailure to stain the upper regions of the small intestine was in starkcontrast with the intense staining in the colon, leading to thespeculation that transporters of the present invention could be used forselective delivery of therapeutics to the colon. Not only were allluminal surfaces of the colon highly fluorescent, but the stainingpattern also revealed that the biotinylated compounds were able topenetrate multiple cell layers and reach vascularized regions of thetissue, suggesting that the transporters should enhance transport intothe bloodstream.

[0461] However, separate studies using monolayers of the colonic cellline, Caco-2, suggests an alternative mechanism. In the Caco-2 systemthe transporters rapidly entered, but did not traverse the monolayer.There are several examples of the transporters exhibiting high rates oftransport into, but limited rates of efflux from cells and tissue. Thisis the case for all suspension cell lines examined to date, the beststudied being the human T cell line, Jurkat. Short oligomers ofD-arginine rapidly enter these cells and exhibit very low exit rates,losing less than 5% of the fluorescent signal after one hour ofincubation at 37° C. An even more relevant example of this phenomenon isthe low levels of CsA measured in the blood stream of mice receivingmultiple topical treatments of a releasable CsA r7 conjugate. As in thecase for the colon, staining patterns in sections of skin treated with abiotinylated analog of the CsA conjugate established that the drugpenetrated into highly vascularized regions of the dermis. In addition,staining with monoclonal antibodies established that one of theprominant cell types in the dermis that were highly fluorescent were theendothelial cells of the capillaries. Nevertheless, detectable levels ofCsA in the blood of these animals were never observed even after tendays of treatment with a 4% ointment applied twice a day. Thisobservation, although anecdotal, is consistent with results betterstudied in this report.

[0462] The ability of the peptides to enter and subsequently exitmultiple layers of cells in the skin, the lungs, and the cardiovascularsystem is in marked contrast to their inability to exit from lymphocytesor Caco-2 cells. Without intending to limit the present invention to aparticular theory or mechanism, one difference is the cells throughwhich the peptides rapidly penetrate are connected by tight junctionsand other membrane structures inherent in tissue architecture, whereasthe individual cells in suspension and perhaps the side of the membraneof endothelial cells contacting the bloodstream lack these features. Ifstructures such as tight or gap junctions modify the surrounding lipidto permit exit of the transporters, then they should be able to diffuserapidly throughout a tissue, such as skin or the colon, but not betransported into the bloodstream. Consistent with this hypothesis is themodel constructed to explain the variations in the rates of appearanceof taxol and CsA in the circulation in this report. In this model, theshort oligomers of arginine greatly promoted uptake into columnarepithelium of the colon or the squamous epithelium of the cheek, but didnot transport the drug into the bloodstream. The blood levels observedwere the result of the diffusion, or active transport, of the drug outof the epithelium into the circulation after hydrolysis of theconjugate.

Example 18 Buccal Delivery of Transporter Conjugates

[0463] Buccal delivery of taxol and CsA involved adding a concentratedsolution (250λ of 5 mg/kg) to the oral cavity of an anesthetized ratlying on its side. Blood samples were taken from the tail vein at timezero (prior to drug injection), and every thirty minutes for theduration of the experiment, which was empirically determined. Clottingwas inhibited by transferring the blood to glass tubes containing 100λof 0.5% EDTA, and the blood was frozen.

[0464] The capacity of the transporters to enter the squamous epitheliallayers of the oral cavity was examined. A mouse was anesthetized, itshead was tipped so that a solution of biotinylated r9 could beadministered. The animal was kept in this position for fifteen minutes,at which time it was sacrificed, and the tongue and cheek weredissected, frozen, sectioned, stained with streptavidin-fluorescein, andcounterstained with propidium iodide. In both the cheek and thetonguethe biotinylated peptides quickly and efficiently penetratedmultiple layers of the epithelia and penetrated deep into the interiorlayers of the tissue, reminiscent of the staining of both the epidermisand dermis of the skin.

[0465] In a second experiment, rats were anesthetized and solutions ofboth taxol and CsA (5 mg/kg) in Cremophor EL:ethanol 1:1 or equimolaramounts of the corresponding r7 conjugates of these drugs in PBS weresimply incubated in the cheek pouch of the animal for the duration ofthe experiment. LC MS was used to determine the blood levels of onlytaxol and the fast releasing r7 conjugate. Both the amount and thekinetics of appearance of taxol in the blood when administered in theoral cavity (FIG. 38) differed from when it was injected into the colon.Buccal administration of taxol conjugates appeared to be less effective,with less than one eighth of the amount of taxol being observed in thecirculation compared with intracolonic injection. Another difference wasthe rate of appearance of the unmodified drug. When administered in theoral cavity, taxol appeared in the blood stream by the first time point,whereas in the colonic injection detectable amounts of taxol did notappear for several hours. Even though there was no difference betweentaxol and the r7-taxol conjugate in the appearance of taxol in thecirculation in buccal administration, approximately twice as muchmaterial reached the circulation when the conjugated was used.

Example 19 Ocular Delivery of Transporter Conjugates

[0466] The ability of the transporters of the invention to penetrate thetissues of the eye was examined. Biotinylated r8 was both injected intothe eyes of rabbits and also applied as eyedrops to the outside of theeye.

[0467] Briefly, 5 drops of a 1 mM solution of biotinylated r8 in PBS wasapplied to both eyes of a rabbit and allowed to incubate 15 minutes. Theanimal was sacrificed and one eye was dissected intact with adjacenttissue, whereas the other was separated into each of its componentparts, frozen and separately sectioned, stained withstreptavidin-fluorescein and counterstained with propidium iodide.results demonstarted staining in the cornea and eyelid, but not thelens. 50 microliters of a 10 mM solution of biotinylated r8 in PBS wasinjected into the vitreus humor of another animal and the animal wassacrificed 30 minutes later and the injected eye was dissected. Againone orbital was frozen intact while the other was dissected and thecomponents separately frozen. Results from the injection experimentsdemonstrated that all interior surfaces of the orb were stained.

Example 20

[0468] This example illustrates the conjugation of cyclosporin to atransport moiety using a pH sensitive linking group (see FIGS. 6A and9B).

[0469] In this example, cyclosporin is converted to its α-chloroacetateester using chloroacetic anhydride to provide 6i (see FIG. 6). The ester6i is then treated with benzylamine to provide 6ii. Reaction of theamine with Boc-protected iminodiacetic acid anhydride provides the acid6iii which is then converted to an activated ester (6iv) with N-hydroxysuccinimide. Coupling of 6iv with L-Arginine heptamer provides theBOC-protected conjugate 6v, which can be converted to conjugate 6vi byremoval of the BOC protecting group according to established methods.

[0470] Transport moieties having arginine groups separated by, forexample, glycine, ε-aminocaproic acid, or γ-aminobutyric acid can beused in place of the arginine heptamer in this and in the followingexamples that show oligoarginine transport groups.

Example 21

[0471] This example illustrates the conjugation of acyclovir to atransport moiety.

[0472] a. Conjugation of Acyclovir to r₇CONH₂

[0473] This example illustrates the conjugation of acyclovir to r₇CONH₂via the linking group:

[0474] A solution of acyclovir (100 mg, 0.44 mmol),dimethylaminopyridine (5.4 mg, 0.044 mmol) and chloroacetic anhydride(226 mg, 1.32 mmol) in dimethylformamide (9 mL) was stirred at roomtemperature for 18 h. The dimethylformamide was removed by evaporation.The crude product was purified by reverse-phase HPLC (22 mm×250 mm C-18column, a 5-25% CH₃CN/H₂O gradient with 0.1% trifluoroacetic acid, 214and 254 nm UV detection) and lyophilized. The product was obtained as awhite powder (62 mg, 47%). ¹H NMR (300 MHz, DMSO-d₆) δ 10.67 (s, 1H),7.88 (s, 1H), 6.53 (s, 1H), 5.27 (s, 2H), 4.35 (s, 2H), 4.21 (t, J=3 Hz,2H), 3.70 (t, J=3 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 168.1, 157.6,154.8, 152.3, 138.6, 117.1, 72.7, 67.1, 65.2, 41.8; TOF-MS (m/z): 302.0[M+H].

[0475] A solution of acyclovir α-chloroester (7 mg, 0.024 mmol),H₂N—C-r7-CONH₂ (50 mg, 0.024 mmol) and diisopropylethylamine (6.4 μL,0.036 mmol) in dimethylformamide (1 mL) was stirred for 18 h. Thedimethylformamide was removed by evaporation. The crude product waspurified by reverse-phase HPLC (22 mm×250 mm C-18 column, a 5-25%CH₃CN/H₂O gradient with 0.1% trifluoroacetic acid, 214 and 254 nm UVdetection) and lyophilized. The desired product was obtained as a whitepowder (24 mg, 69%). TOF-MS (m/z): 494.6 [(M+H)/3], 371.0 [(M+H)/4].

[0476] The yield could be increased by using 10 molar equivalents ofdiisopropylethylamine rather than 1.5 molar equivalents. Product wasagain obtained as a white powder (79%). TOF-MS (m/z): 508.7 [(M+H)/3],381.5 [(M+H)/4], 305.5 [(M+H)/5].

[0477] Reactions were carried out as illustrated above, using thesynthetic techniques provided in the examples above.

[0478] i) Biotin-aminocaproic acid-r5-Cys(acyclovir)-CONH₂ was obtainedas a white powder (36%). TOF-MS (m/z): 868.2 {(M+2 TFA)/2], 811.2 [(M+1TFA)/2], 754.1 [(M+1 TFA)/3], 503.0 [(M+H)/3], 377.4 [(M+H)/4].

[0479] Similarly,

[0480] ii) Biotin-aminocaproic acid-r7-C(acyclovir)-CONH₂— was obtainedas a white powder (33%). TOF-MS (m/z):722.1 [(M+3 TFA)/3], 684.6 [(M+2TFA)/3], 607.1 [(M+H)/3], 455.5 [(M+H)/4], 364.8 [(M+H)/5], 304.3[(M+H)/6].

Example 22

[0481] This example illustrates the conjugation of hydrocortisone to atransport moiety.

[0482] a. Conjugation of Hydrocortisone to r₇CONH₂

[0483] To a solution of hydrocortisone (500 mg, 1.38 mmol), scandiumtriflate (408 mg, 0.83 mmol) and chloroacetic anhydride (708 mg, 4.14mmol) in dry THF was added dimethylaminopyridine (506 mg, 4.14 mmol).The solution turned bright yellow upon addition ofdimethylaminopyridine. After 30 min the solvent was evaporated off andthe crude material taken up into ethyl acetate (100 mL). The ethylacetate layer was washed with 1.0 N HCl and brine. The organic phase wascollected, dried (Na₂SO₄) and evaporated to provide the product as awhite solid (533 mg, 88%). ¹H NMR (300 MHz, DMSO-d₆) δ 5.56 (s, 1H),5.46 (s, 1H), 5.20 (d, J=18 Hz, 1H), 4.85 (d, J=18 Hz, 1H), 4.51 (s,2H), 4.37 (br s, 1H), 4.27 (br s, 1H), 2.54-2.33 (m, 2H), 2.22-2.03 (m,3H), 1.99-1.61 (m, 8H), 1.52-1.24 (m, 5H), 1.02-0.98 (d, J=12 Hz, 1H),0.88-0.85 (d, J=9 Hz, 1H), 0.77 (s, 3 H); ¹³C NMR (75 MHz, DMSO-d₆) δ205.4, 198.8, 173.0, 167.6, 122.3, 89.5, 69.7, 67.3, 56.4, 52.4, 47.8,41.6, 39.7, 35.0, 34.3, 34.0, 33.6, 32.3, 32.0, 24.2, 21.3, 17.4; TOF-MS(m/z): 439.1 (M+H).

[0484] (Reference for acetylation—Zhao, H.; Pendri, A.; Greenwald, R. B.J. Org. Chem. 1998, 63, 7559-7562.)

[0485] A solution of hydrocortisone α-chloroester (31 mg, 0.071 mmol),H₂N—C-r7-CONH₂ (150 mg, 0.071 mmol) and diisopropylethylamine (15 μL,0.085 mmol) in dimethylformamide (1 mL) was stirred for 18 h. Thedimethylformamide was evaporated off. The crude product purified byreverse-phase HPLC (22 mm×250 mm C-18 column, a 5-30% CH₃CN/H₂O gradientwith 0.1% trifluoroacetic acid, 214 and 254 nm UV detection) andlyophilized. The desired product was obtained as a white powder (25 mg,14%). TOF-MS (m/z): 1037.4 [(M+4 TFA)/2], 616.1 [(M+2 TFA)/3], 578.3[(M+1 TFA)/3], 540.5 [(M+H)/3], 405.7 [(M+H)/4], 324.5 [(M+H)/5].

[0486] The use of 10 molar equivalents of diisopropylethylamine ratherthan 1.2 molar equivalents provided the desired product as a yellowpowder (52% yield). TOF-MS (m/z): 887.0 [(M+1TFA)/2], 830.6 [(M+H)/2],553.7 [(M+H)/3], 415.5 [(M+H)/4].

[0487] Reactions were carried out as illustrated above, using thesynthetic techniques provided in the examples above.

[0488] i) Biotin-aminocaproic acid-r5-C(hydrocortisone)-CONH₂—Used 10molar equivalents of diisopropylethylamine rather than 1.2 molarequivalents. Product a white powder (65%). TOF-MS (m/z): 880.7 [(M+1TFA)/2], 548.7 [(M+H)/3].

[0489] ii) Biotin-aminocaproic acid-r7-C(hydrocortisone)-CONH₂—Used 10molar equivalents of diisopropylethylamine rather than 1.2 molarequivalents. Product a white powder (36%). TOF-MS (m/z): 692.3 [(M+1TFA)/3], 652.8 [(M+H)/3], 520.0 [(M+1 TFA)/4], 490.0 [(M+H)/4], 392.5[(M+H)/5].

Example 23

[0490] This example illustrates the conjugation of taxol to a transportmoiety.

[0491] a. Conjugation of Taxol to r₇-CONH₂

[0492] This example illustrates the application of methodology outlinedabove to the preparation of a taxol conjugate (see FIG. 12).

[0493] Taxol was treated with α-chloro acetic anhydride providing theC-2′ chloro acetyl derivative 12i in essentially quantitative yield.

[0494] The halogen atom of the chloroacetate ester was displaced by thethiol of an N-tenninal (L) cysteine containing heptamer of arginine. Toavoid degradation of the transporter entity by proteases in-vivo,D-arginine was used as the building unit. Conjugation reactions wereperformed at room temperature in DMF in the presence ofdiisopropylethylamine. The final products were isolated by RP-HPLC andlyophilized to white powders. It is important to note that the nativeconjugate (R=H) is isolated as its TFA salt at the cysteine primaryamine. The conjugates are generally quite hygroscopic and readilydissolve in water.

[0495] The conjugate wherein R=H was designed to release the parent drugvia a nucleophilic attack of the N-terminal nitrogen onto the C2′ estercarbonyl. The protonation state of this nitrogen is crucial for thismechanism, since only the free amine will be capable of this release.Additionally, both conjugates share a common α-hetero atom substitutedacetate moiety making them susceptible to simple ester hydrolysis. Thisoffers an additional release pathway.

Example 24

[0496] This example illustrates two methods of linking active agents totransport moieties. Illustration is provided for retinoic acidderivatives linked to poly-D-Arg derivatives but can be applied tolinkages between other biological agents and the transport moieties ofthe present invention.

[0497] a. Linkage Between a Biological Agent Having an AldehydeFunctional Group

[0498] This example illustrates the preparation of a conjugate between anonamer of D-arginine (H₂N-r₉-CO₂H.10TFA) and either all trans-retinalor 13-cis-retinal. FIG. 40 provides a schematic presentation of thereactions. As seen in FIG. 40, condensation of either retinal withH₂N-r₉-CO₂H.10TFA in MeOH in the presence of 4 Å molecular seives atroom temperature for four hours results in the formation of a Schiffbase-type linkage between the retinal aldehyde and the amino terminalgroup. Purification of the conjugate can be accomplished by filteringthe molecular sieves and removing methanol under reduced pressure.

[0499] b. Conjugation of Retinoic Acid to r₇-CONH₂

[0500] This example illustrates the preparation of a conjugate betweenretinoic acid and r₇-CONH₂ using the linking group

[0501] Here, preparation of the conjugate follows the scheme outlined inFIG. 41. In this scheme, retinoic acid (41ii) is first combined with thechloroacetate ester of 4-bydroxymethyl-2,6-dimethylphenol (41i) toprovide the conjugate shown as 41iii. Combination of 41i with retinoicacid in methylene chloride in the presence of dicyclohexylcarbodiimideand a catalytic amount of 4-dimethylaminopyridine provided the retinoidderivative 41iii in 52-57% yield. Condensation of 41iii withH₂NCys-r₇CONH₂.8TFA in the presence of diisopropylethylamine (DMF, roomtemperature, 2 h) provides the desired conjugated product 41iv.

[0502] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

What is claimed is:
 1. A method of targeting a compound to agastrointestinal epithelium of an animal, the method comprisingadministering to the gastrointestinal epithelium a conjugate comprisingthe compound and a delivery-enhancing transporter, wherein: i. thecompound is attached to the delivery-enhancing transporter through alinker; and ii. the delivery-enhancing transporter comprises fewer than50 subunits and comprises at least 5 guanidino or amidino moieties,thereby increasing delivery of the conjugate into the gastrointestinalepithelium compared to delivery of the compound in the absence of thedelivery-enhancing transporter.
 2. The method of claim 1, whereindelivery of the conjugate into the gastrointestinal epithelium isincreased at least two-fold compared to delivery of the compound in theabsence of the delivery-enhancing transporter.
 3. The method of claim 1,wherein delivery of the conjugate into the gastrointestinal epitheliumis increased at least ten-fold compared to delivery of the compound inthe absence of the delivery-enhancing transporter.
 4. The method ofclaim 1, wherein the linker is a releasable linker.
 5. The method ofclaim 1, wherein the subunits are amino acids.
 6. The method of claim 1,wherein the conjugate has a structure selected from the group consistingof structures 3, 4, or 5, as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R² is hydrogen, alkyl, aryl, acyl, orallyl; R³ comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n is 1to
 10. 7. The method of claim 6, wherein X is selected from the groupconsisting of —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate phosphinate, andCR⁷R⁸, wherein R⁷ and R⁸ are each independently selected from the groupconsisting of H and alkyl.
 8. The method of claim 6, wherein theconjugate comprises structure 3, Y is N, and R² is methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.
 9. The method of claim 6,wherein R² is benzyl; k, m, and n are each 1, and X is —OC(O)—.
 10. Themethod of claim 6, wherein the conjugate comprises structure 4; R⁴ is S;R⁵ is NHR⁶; and R⁶ is hydrogen, methyl, allyl, butyl or phenyl.
 11. Themethod of claim 6 wherein the conjugate comprises structure 4; R⁵ isNHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl; and k and m areeach
 1. 12. The method of claim 1, wherein the conjugate comprisesstructure 6 as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or para configuration, which aryl group can besubstituted or unsubstituted; R³ comprises the delivery-enhancingtransporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and
 2. 13. The method of claim 12, whereinX is selected from the group consisting of —C(O)O—, —C(O)NH—, —OC(O)NH—,—S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—, —SONH—, phosphate,phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸ are eachindependently selected from the group consisting of H and alkyl.
 14. Themethod of claim 12, wherein R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen,methyl, allyl, butyl or phenyl.
 15. The method of claim 1, wherein theconjugate comprises at least two delivery-enhancing transporters. 16.The method of claim 1, wherein the conjugate is administered buccally.17. The method of claim 1, wherein the conjugate is administered as asuppository.
 18. The method of claim 1, wherein the delivery-enhancingtransporter comprises a non-peptide backbone.
 19. The method of claim 1,wherein the delivery-enhancing transporter is not attached to an aminoacid sequence to which the delivery enhancing transporter molecule isattached in a naturally occurring protein.
 20. The method of claim 1,wherein the delivery-enhancing transporter comprises from 5 to 25guanidino or amidino moieties.
 21. The method of claim 20, wherein thedelivery-enhancing transporter comprises between 7 and 15 guanidinomoieties.
 22. The method of claim 20, wherein the delivery-enhancingtransporter comprises at least 6 contiguous guanidino and/or amidinomoieties.
 23. The method of claim 1, wherein the delivery-enhancingtransporter consists essentially of 5 to 50 amino acids, at least 50percent of which amino acids are arginines or analogs thereof.
 24. Themethod of claim 23, wherein the delivery-enhancing transporter comprises5 to 25 arginine residues or analogs thereof.
 25. The method of claim24, wherein at least one arginine is a D-arginine.
 26. The method ofclaim 25, wherein all of the arginines are D-arginines.
 27. The methodof claim 23, wherein at least 70 percent of the amino acids thatcomprise the delivery-enhancing transporter are arginines or arginineanalogs.
 28. The method of claim 23, wherein the delivery-enhancingtransporter is seven contiguous D-arginines.
 29. The method of claim 1,wherein the compound is a therapeutic for the disease selected from thegroup consisting of inflammatory bowel disease, colon cancer, ulcerativecolitis, gastrointestinal ulcers, constipation and imbalance of salt andwater absorption.
 30. The method of claim 1, wherein the compound isselected from the group consisting of immunosuppressives, ascomycins,corticosteroids, laxatives, antibiotics and anti-neoplastic agents. 31.The method of claim 1, wherein the compound is targeted to the iliemand/or colon.
 32. A method for enhancing delivery of a compound into andacross one or more layers of an animal ocular epithelial tissue, themethod comprising: administering to the ocular epithelial tissue aconjugate comprising the compound and a delivery-enhancing transporter,wherein: i. the compound is attached to the delivery-enhancingtransporter through a linker, and ii. the delivery-enhancing transportercomprises fewer than 50 subunits and comprises at least 5 guanidino oramidino moieties, thereby increasing delivery of the conjugate into thegastrointestinal epithelium compared to delivery of the compound in theabsence of the delivery-enhancing transporter.
 33. The method of claim32, wherein delivery of the conjugate into the gastrointestinalepithelium is increased at least two-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter.
 34. Themethod of claim 32, wherein delivery of the conjugate into thegastrointestinal epithelium is increased at least ten-fold compared todelivery of the compound in the absence of the delivery-enhancingtransporter.
 35. The method of claim 32, wherein the linker is areleasable linker.
 36. The method of claim 32, wherein the subunits areamino acids.
 37. The method of claim 32, wherein the conjugate has astructure selected from the group consisting of structures 3, 4, or 5,as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R² is hydrogen, alkyl, aryl, acyl, orallyl; R³ comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n is 1to
 10. 38. The method of claim 37, wherein X is selected from the groupconsisting of —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate phosphinate, andCR⁷R⁸, wherein R⁷ and R8 are each independently selected from the groupconsisting of H and alkyl.
 39. The method of claim 37, wherein theconjugate comprises structure 3, Y is N, and R² is methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.
 40. The method of claim 37,wherein R² is benzyl; k, m, and n are each 1, and X is —OC(O)—.
 41. Themethod of claim 37, wherein the conjugate comprises structure 4; R⁴ isS; R⁵ is NHR⁶; and R⁶ is hydrogen, methyl, allyl, butyl or phenyl. 42.The method of claim 37, wherein the conjugate comprises structure 4; R⁵is NHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl; and k and m areeach
 1. 43. The method of claim 32, wherein the conjugate comprisesstructure 6 as follows:

wherein: R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or para configuration, which aryl group can besubstituted or unsubstituted; R³ comprises the delivery-enhancingtransporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and
 2. 44. The method of claim 43, whereinX is selected from the group consisting of —C(O)O—, —C(O)NH—, —OC(O)NH—,—S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—, —SONH—, phosphate,phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸ are eachindependently selected from the group consisting of H and alkyl.
 45. Themethod of claim 43, wherein R₄ is S; R⁵ is NHR⁶; and R⁶ is hydrogen,methyl, allyl, butyl or phenyl.
 46. The method of claim 32, wherein theconjugate comprises at least two delivery-enhancing transporters. 47.The method of claim 32, wherein the conjugate is administered as an eyedrop.
 48. The method of claim 32, wherein the conjugate is administeredas an injection
 49. The method of claim 32, wherein thedelivery-enhancing transporter comprises a non-peptide backbone.
 50. Themethod of claim 32, wherein the delivery-enhancing transporter is notattached to an amino acid sequence to which the delivery enhancingtransporter molecule is attached in a naturally occurring protein. 51.The method of claim 32, wherein the delivery-enhancing transportercomprises from 5 to 25 guanidino or amidino moieties.
 52. The method ofclaim 51, wherein the delivery-enhancing transporter comprises between 7and 15 guanidino moieties.
 53. The method of claim 51, wherein thedelivery-enhancing transporter comprises at least 6 contiguous guanidinoand/or amidino moieties.
 54. The method of claim 32, wherein thedelivery-enhancing transporter consists essentially of 5 to 50 aminoacids, at least 50 percent of which amino acids are arginines or analogsthereof.
 55. The method of claim 54, wherein the delivery-enhancingtransporter comprises 5 to 25 arginine residues or analogs thereof. 56.The method of claim 55, wherein at least one arginine is a D-arginine.57. The method of claim 56, wherein all of the arginines areD-arginines.
 58. The method of claim 54, wherein at least 70 percent ofthe amino acids that comprise the delivery-enhancing transporter arearginines or arginine analogs.
 59. The method of claim 54, wherein thedelivery-enhancing transporter is seven contiguous D-arginines.
 60. Themethod of claim 32, wherein the compound is a therapeutic for thedisease selected from the group consisting of conjunctivitis, bacterialinfections, viral infections, dry eye, and glaucoma.
 61. The method ofclaim 32, wherein the compound is selected from the group consisting ofantibacterial compounds, antiviral compounds, cyclosporin, ascomycins,and corticosteroids.